In recent years, percutaneous catheter interventions have continuously evolved, becoming an essential strategy for interventional diagnosis and treatment of many structural heart diseases and arrhythmias. Along with the increasing complexity of cardiac interventions comes ever more complex demands for intraoperative imaging. Intracardiac echocardiography (ICE) is well-suited for these requirements with real-time imaging, real-time monitoring for intraoperative complications, and a well-tolerated procedure. As a result, ICE is increasingly used many types of cardiac interventions. Given the lack of relevant guidelines at home and abroad and to promote and standardize the clinical applications of ICE, the members of this panel extensively evaluated relevant research findings, and they developed this consensus document after discussions and correlation with front-line clinical work experience, aiming to provide guidance for clinicians and to further improve interventional cardiovascular diagnosis and treatment procedures. The consensus was written by the “Chinese ICE Expert Group,” which includes experts in echocardiography, cardiac electrophysiology, congenital heart disease, valvular heart disease and so on. The consensus was initiated by Professor Zhang Yun and Ma Changsheng, and Professor Zhong Jingquan and Long Deyong were mainly responsible for the completion of the manuscript. We held three relevant meetings  regarding the consensus. Details of meetings are provided in the Intracardiac echocardiography (ICE) is an ultrasound imaging technique able to perform real-time high-quality imaging and/or hemodynamic measurement of the heart and its adjacent tissues with an ultrasound probe placed at the tip of a catheter delivered into the cardiac chambers via peripheral vessels –3. In viA catheter with an ultrasound transducer was first delivered into the cardiac chambers of a dog via the jugular vein to obtain endocardial echo images of the left and right ventricles as early as 1960 . Since tIntracardiac echocardiography catheters currently in use can be classified into the following two types by their technologic principles: the mechanical rotary ultrasound catheter and the phased array ultrasound catheter . The forInitially, ICE was mainly used to guide the interventional closure of atrial septal defect (ASD) and patent foramen ovale (PFO), with comparable image quality to that of transesophageal echocardiography (TEE). Nowadays, ICE is more common imaging modality used during these procedures, as it requires neither general anesthesia nor sonographer assistance , 13. In Early use of ICE was associated with a complication incidence of approximately 4%, mainly atrial tachycardia induced by the catheter manipulation in the right atrium . DespiteIn conclusion, with the ability to image intracardiac structures and the adjacent anatomical relationship in real-time, ICE has gradually increased in use to guide interventional procedures for multiple structural heart diseases and arrhythmias and monitor intraoperative complications. Recommendations for the clinical application of ICE are shown in Intracardiac echocardiography helps the operator understand the key anatomy associated with arrhythmias, determine the spatial relationship between mapping and ablation catheters and their corresponding cardiac structures, and directly observe and guide the adjustment of degree of contact between the tip of the ablation catheter and the tissue. ICE can be used to monitor the formation, site, extent, and degree of ablation lesions to help determine the efficacy of ablation. Moreover, ICE can be used to monitor for complications in real-time to help determine their sites and severity. With the real-time monitoring function of ICE, complications can often be detected and managed before a hemodynamic change occurs. ICE can visualize the entire cardiac structure and accurately locate the aortic root and pulmonary sinus even when manipulated in the right heart. ICE is moreover instructive during ablation of arrhythmias originating from the outflow tracts, and it is critical for mapping and ablation of arrhythmias originating from protruding intracardiac structures, such as papillary muscle, false tendon, and moderator band. Furthermore, ICE allows observation of myocardial contraction, ventricular arrhythmia substrates such as scar/fibrosis, all the while enabling reduction of exposure to X-ray radiation and contrast agents.During interventional procedures for atrial arrhythmias, ICE can aid in the assessment of anatomical characteristics of the pulmonary veins , the guidance of transseptal puncture, the screening for atrial/atrial appendage thrombus, and the monitoring of ablation lesion formation , 20. ICEIntracardiac echocardiography and TEE have distinct advantages and limitations. TEE has traditionally been the gold standard for the exclusion of left atrial and left atrial appendage thrombi and for anatomic delineation during catheter ablation for atrial fibrillation and LAAC. It is however, associated with increased patient discomfort and risk. Patients need to fast prior to the examination, and they need to be highly cooperative during the examination, and there is risk of esophageal injury during the examination. Left atrial CTA may also be used for the exclusion of left atrial appendage thrombi, but limitations include relatively high false-positive rates and the need for institutional experience for high quality images and accurate interpretation. IV contrast agent injection is required, with associated risks of anaphylaxis and kidney injury. Compared with TEE, ICE is expensive, as it uses a disposable catheter, but it is associated with less discomfort, greater compliance, lower incidence of complications, and less overall procedural radiation exposure. Many clinical studies evaluation ICE for left atrial and left atrial appendage thrombi have shown that –29. ICE Intracardiac echocardiography is a good option for the screening of left atrial and left atrial appendage thrombi in patients unable or unwilling to undergo TEE due to esophageal pathology, comorbidities rendering repeated sedation events riskier, or if left atrial CTA cannot confirm or rule out left atrial appendage thrombus. Further, ICE in patients who have undergone TEE or CTA, especially for when TEE suggests of “significant clouding,” “suspected thrombus,” and other unclear findings ICE may add additional diagnostic value , 31.Transseptal puncture was first used for left atrial manometry by Ross et al.  in 1959 Using ICE as the primary tool to guide transseptal puncture can make the puncture process easier, safer, and more specifically directed within the interatrial septum. Unlike TEE, ICE can be used in combination with the mapping system for 3D reconstruction by a single operator, with a wider field of view and no need for general anesthesia. One particular ICE system  enables contouring the cardiac structures visualized on ICE onto the EA map, as a magnet-enabled ICE catheter tip allows orientation of the catheter and hence ICE images within the 3D map. Before the transseptal puncture is performed, the operator can reconstruct key structures including the left atrium, fossa ovalis and aorta using ICE, and then select and mark the appropriate puncture site by adjusting the image sector as needed for subsequent steps. For the actual puncture procedure, ICE can visualize the entire TSP process. As a key step, when the puncture needle sheath is delivered into the fossa ovalis, a “tenting sign” will be observed at the fossa ovalis by ICE. Microbubbles seen using saline injection after the needle is inserted can help to further confirm the needle tip location in relation to the fossa ovalis. Proper entry of the needle tip into the left atrium can be confirmed by microbubble shadowing in the left atrium during saline injection through the needle , there is often a spatial deviation between the planned ablation point and the real point. This may be caused by shifts in patient respiratory movement during the procedure, mapping inaccuracies, or anatomic-spatial changes resulting from changes in rhythm. However, with ICE two-dimensional imaging in combination with the Carto-Sound module to construct three-dimensional models of the left atrium and pulmonary veins in a 3D electroanatomical mapping system, the accuracy of FAM can be improved. In most clinical studies, the ultrasound catheter was placed in the right atrium. To further improve the model accuracy, the ICE catheter was then placed in the left atrium in some studies. In this way, the modeling was not only feasible but also more accurate (compared with modeling in the right atrium) . On thisReal-time three-dimensional ICE, or four-dimensional ICE, is a new technique emerging in recent years. Few studies have been reported on ICE-guided catheter ablation for atrial fibrillation. However, with the conduct of relevant research, we expect it to be useful in the whole process of catheter ablation for atrial fibrillation with improved efficiency and safety  is the standard approach for treating atrial fibrillation, restoring and maintaining sinus rhythm . CryobalPulmonary vein isolation alone demonstrates a low success rate in patients with persistent atrial fibrillation. Given this, some studies reported the application of CBA for PVI with additional substrate ablation in the treatment of persistent atrial fibrillation in recent years: (1) left atrial roof linear ablation ; (2) lefRapid diagnosis and prevention of potential complications during ablation is one of the most important functions of ICE . A recenThe application of ICE in catheter ablation can reduce the incidence of perioperative complications, especially the incidence of serious complications such as thromboembolism and cardiac tamponade , 55–64, Acute cardiac tamponade is one of the most common serious complications associated with catheter manipulation during ablation. ICE can detect early pericardial effusion along the lower ventricular border and posterior left atrium, which can be managed by reversing anticoagulant therapy to prevent cardiac tamponade , 62, 63.Thromboembolism is another serious complication associated with left cardiac ablation. ICE-guided catheter ablation allows real-time observation of the factors associated with increased stroke risk, such as thrombosis at the catheter, sheath, and endocardial lesion sites and clot formation on the ablation electrodes . Once a Esophageal injury and atrio-esophageal fistula are important issues in atrial fibrillation ablation. The incidence of the atrio-esophageal fistula is 0.05–2%, while the esophageal injury is still common . The abiPulmonary vein stenosis is associated with ablation sites at the pulmonary vein antrum, likely when lesions are delivered in a more ostial location. This complication can be mitigated or avoided by accurately localizing the optimal ablation site with ICE , 61. ICEComplications associated with ICE application itself are rare. However, as the ICE catheter is relatively stiff and may result in vascular injury and/or perforation during its advancement, it should be advanced with care .The ability to accurately and clearly delineate cardiac anatomy is directly related to procedural efficiency, efficacy, and safety. False lumens and other anatomic inaccuracies are inevitable with conventional contact-type three-dimensional reconstruction, affecting the operators’ judgment of ablation targets or special structures. ICE, as a non-contact three-dimensional reconstruction technique, is not limited by catheter position. With ICE, the atrial body, pulmonary veins, atrial appendages and other structures can be entirely reconstructed in an objective and accurate manner through simple sector adjustments. Furthermore, based on the integration of ICE and contact-type reconstruction of key structures, a more realistic and accurate anatomical model can be obtained after image fusion, creating an anatomic framework for subsequent ablation .During ablation, ICE can not only track the ablation catheter in real time and identify the catheter position in complex structures such as the left atrial appendage-pulmonary vein ridge, but it can also help avoid esophageal injury and excessive ablation at thinner parts of the myocardium.Intracardiac echocardiography-guided standardized approach to ablation procedures may improve several procedural outcomes. Beginners can obtain satisfactory ablation results with ICE-guided low X-ray catheter ablation for atrial fibrillation, with an average fluoroscopy time of 2.3 ± 3.0 min. The learning curve is short, with the fluoroscopy time dropping rapidly to 9 s from 3.8 min during a study evaluating learning curve .Experts’ recommendation: (1) If possible, ICE-guided transseptal puncture is recommended, especially for patients with abnormal interatrial septal anatomy. (2) Routine TEE or left atrial and pulmonary vein CT examination should be performed before catheter ablation in patients with atrial fibrillation to rule out left atrial appendage thrombus and preliminarily assess the shape and size of the left atrial appendage; for patients with suspected thrombus that is difficult to distinguish preoperatively or unable to tolerate and unable to undergo left atrial and pulmonary vein CT or TEE examination, ICE can be applied intraoperatively to provide additional assessment for left atrial appendage thrombus and re-assess the shape and size of the left atrial appendage; meanwhile, ICE can also be used as an alternative monitoring and assessment technique in patients intolerable to TEE. (3) If possible, ICE-guided catheter ablation requiring low X-ray or zero X-ray is recommended for atrial fibrillation in medical centers to reduce the radiation exposure of patients and surgeons. (4) Imaging and blood flow monitoring by ICE may help to guide cryoballoon positioning and improve the efficiency and efficacy of ablation. (5) Catheter ablation for patients who are not suitable for radiation including pregnant women should be performed under ICE guidance. (6) During the training of new electrophysiologists, instruction in ICE is recommended.Intracardiac echocardiography plays an important role in the catheter ablation for ventricular arrhythmias, just as in interventions for atrial arrhythmias. It is mainly used to monitor cardiac structures in real-time, reduce the duration of surgery and radiation exposure , delineaLeft posterior fascicular ventricular tachycardia (VT) is the most common type of idiopathic ventricular tachycardia, and its electrophysiological mechanism remains controversial. It is traditionally considered to be a macro-reentrant arrhythmia originating from the left posterior fascicle, which needs to be differentiated from the ventricular tachycardia originating from papillary muscles in clinical practice. However, definitive differentiation between the two based on ECG and EP is sometimes difficult. In fact, it has been proved that the mechanism of a part of fascicular ventricular tachycardia is closely related to such structures as Purkinje fibers and false tendons around the papillary muscles . For thiIdiopathic outflow tract ventricular arrhythmias mainly include monomorphic ventricular premature beats, non-sustained ventricular tachycardia, and sustained monomorphic ventricular tachycardia. Right ventricular outflow tract ventricular arrhythmia is the most common type of ventricular arrhythmias in clinical practice, mostly idiopathic, accounting for about 80% of outflow tract ventricular arrhythmias . In receThe right ventricular outflow tract myocardial tissue anatomically extends to the pulmonary valve and pulmonary artery, making the positioning of ablation targets more complex. Some cases failing with subvalvular ablation may be successfully treated by supravalvular ablation. However, for supravalvular ablation, a transvalvular approach may lead to valve injury and other complications. In this case, the application of ICE not only helps to avoid such complications, but also enables sound reconstruction and real-time monitoring of the pulmonary artery, aortic valve, left anterior descending artery and right ventricular outflow tract during the surgery, and avoids the use of fluoroscopy and contrast agent and the occurrence of such complications as valvular insufficiency. With ICE placed in the right atrial appendage to obtain real-time cross-section images of pulmonary valves , it is eGiven the complex anatomy  adjacent to the right ventricular outflow tract, RF energy transmitted close to the coronary arteries may cause obstruction of major epicardial vessels  and possibly myocardial infarction. It is therefore essential to identify the anatomic location of these structures, traditionally using coronary angiography. ICE enables accurate reconstruction and real-time dynamic observation of proximal arterial anatomy during ablation, avoiding the use of contrast agents and further reducing risk to patients. Right ventricular outflow tract ventricular arrhythmias usually originate from the root of the pulmonary artery. Without ICE, operators may be unable to determine the relationship between the ablation target and a specific anatomical structure at that site due to trabecular muscles and fibrous tissues arranged in a crisscross pattern at this site, the low resolution of 3D navigation and the 2D image overlay of fluoroscopy/radiography. However, ICE can visualize fine anatomical landmarks of the heart in real time and guide the catheter operation  throughoWith improved understanding and experience, the recognized incidence of ventricular arrhythmia originating from the left ventricular outflow tract is increasing year by year, especially ventricular premature beat/ventricular tachycardia originating from the aortic sinus and its adjacent areas. As the aortic sinus is located in the central part of the heart and with adjacent tissues are critical and anatomically complex, patients whose ventricular arrhythmias originate from these anatomically complex regions  and cardiac CT/MRI. Contours of the left (or right) ventricle and associated structures can be reconstructed with ICE via the right ventricle or sometimes the right atrium or CS. ICE can localize the catheter in relationship with the ventricular tissues, resulting in likely lowered risk of cardiac perforation due to excessive contact force. Especially in ischemic cardiomyopathy patients with ventricular aneurysm, left ventricular anatomy is often distorted, so that ICE reconstruction of a ventricular aneurysm and the aneurysmal neck can be helpful to define the anatomy and hence potential ablation target  Figure .Another important advantage of ICE for such patients is the delineation and quantification of the scar area. The scar area appears as a hyperechoic area on the ultrasound sector, and the marginal area appears as a mixture of medium to high echo densities, which is significantly different from that of normal ventricular myocardium . A studyExperts’ recommendation: (1) As a safe, effective, efficient, and comprehensive approach with unique advantages in identifying and locating ablation targets and specific cardiac anatomy, and with superiority to other techniques in identifying small abnormal foci, ICE is recommended as an imaging modality in radiofrequency ablation of left and right ventricular outflow tract arrhythmias, post-TAVR ventricular tachycardia and VSD-induced ventricular tachycardia in adults. Radiofrequency ablation of ventricular tachycardia augmented by ICE may also reduce readmission rates, the possibility of repeated ablation, and the incidence of complications. (2) ICE can be actively applied in ventricular arrhythmia patients with ventricular wall dysfunction who require ablation.Intracardiac echocardiography is now the most widely used technique in ASD and PFO closure among the interventional closure treatments for various congenital heart diseases. Statistical results in the United States indicate that the use of ICE in ASD closure has increased from an initial 9.7% to more than 50% today . In the Intracardiac echocardiography has a higher image resolution than TEE. Despite the lack of multiplanar imaging capabilities, it can still image the interatrial septum from multiple views with its flexible probe, thereby obtaining images similar to or better than those obtained by TEE . ICE canAnother significant advantage of ICE is that it can significantly shorten intraoperative X-ray exposure time , effectiCost is one of the major factors restricting the wide application of ICE in clinical practice. ICE itself is more expensive than TEE, but if ICE is used, general anesthesia may be exempted and the average hospital stay can be shortened , 105, thCurrently, there is relatively little experience with ICE for PDA and VSD procedures. Percutaneous interventional PDA closure is currently the standard of care for PDA, and conventional therapy is aortography-guided interventional closure. However, for most patients with PDA, a large amount of contrast agent is required in aortography, and in some cases, images obtained are not clear enough to assess PDA anatomy , 107, soIn the percutaneous closure of membranous VSD, TEE plays an important role in the anatomical assessment of the defect and the surgeon’s intraoperative decision. However, due to the long duration of VSD closure, TEE related examinations should be performed under general anesthesia. ICE provides clear images of the membrane of VSD, and its measurement results are similar to those obtained by TEE; in addition, ICE and TEE are comparable in judging the relationship between the defect and the aortic valve and tricuspid valve, measuring the size of the defect and giving guidance at different stages.Experts’ recommendation: (1) ICE performs well in anatomical measurements of relevant structures, real-time guidance of closure device implantation, assessment of post-closure residual shunt, etc. If possible, medical centers should apply ICE to guide the interventional procedure of secondary ASD and PFO; (2) ICE is recommended to guide the interventional closure in patients with complex or special ASD and PFO, especially the closure in patients with complex ASD, intolerant to TEE, with impaired left ventricular systolic function and unable to receive fluoroscopy, especially children, pregnant women, obese patients; (3) ICE is recommended to guide the interventional closure of PDA in patients with large PDA, renal insufficiency, and contrast agent allergy; (4) ICE is recommended to guide the closure in VSD patients with complex anatomy.Severe symptomatic aortic valve stenosis is a life-threatening disease with a 2-year mortality rate as high as 50% , and TAVKadakia reported a TAVR case  that wasIntraoperative pressure gradients were underestimated by TEE compared with conventional measurements, but not by ICE. Both ICE and TEE detected new intracardiac thrombi. In this study, the authors concluded that ICE was compatible with sedation and local anesthesia and could be used for guidance in place of TEE. Also, it appeared to better match the operational flow during TAVR than TEE. In a study of 21 patients scheduled for TAVR, the major intraoperative imaging modality was 3D ICE . These pPara-valvular leak is one of the most common complications of TAVR. A case of PVL on postoperative echocardiography and cardiovascular imaging was described . EccentrAs for the operation of the ICE catheter during the TAVR procedure, one should advance the 8-Fr AcuNav™ catheter to the superior vena cava from the right internal jugular vein and rotate the catheter counterclockwise to obtain images of the ascending aorta and assess its anatomy preoperatively. Images of the interatrial septum can be obtained when the catheter is advanced a few centimeters further to judge whether there is ASD or PFO. Counterclockwise rotation is continued to obtain long-axis views of the right atrium, tricuspid valve, and right ventricle, where the preoperative TR and estimated right ventricular systolic pressure can be measured. When the catheter is advanced counterclockwise with forward flexion in the right ventricle, the long-axis view of the left ventricle displays left ventricular contraction and pericardial effusion, and this position can be maintained during much of the operation. When the catheter is returned clockwise and pulled to the right atrium, the long-axis view of the aortic valve is obtained, where the preoperative aortic valve velocity and the diameter of the aortic valve complex can be measured. After TAVR, evaluation for complications such as PVL from the margin of the non-coronary valve leaflet can be performed .There are limitations associated with ICE, including the need for additional venous access, the learning curve related to new devices, and the possible increased cost . SpecialExperts’ recommendations: (1) ICE is a recommended alternative to TEE for TAVR in elderly patients with aortic valve stenosis who have esophageal lesions or are not suitable for general anesthesia; (2) ICE is equivalent to CT 3D reconstruction in the measurement of aortic valve and supravalvular and subvalvular structures and detection of possible complications during and after operation, and even superior to TEE in some cases; (3) It is suitable for surgeons experienced in both TAVR and ICE , and Transcatheter Mitral Valve Replacement (TMVR). ICE-guided transseptal puncture, ICE catheter access to the left atrium, and imaging and functional assessment of the mitral valve are fundamental operations of these therapeutic approaches, and we will uniformly describe them in detail. Additional procedures with different approaches are described in the corresponding sections.Transseptal puncture is a key step in mitral valve intervention, and it directly affects the success rate of mitral valve intervention. Under fluoroscopic guidance, deliver the ICE probe to the inferior position of the right atrium via the femoral vein, i.e., at the level of the tricuspid annulus, and appropriately rotate the catheter, until the right atrium, tricuspid valve, right ventricle, and right ventricular outflow tract can be visualized (home view). At this point, the posterior leaflet of the tricuspid valve is usually in the direction of 9 o’clock, while the anterior leaflet  is in the direction of 3 o’clock. This is the most fundamental view of ICE. Under fluoroscopy, deliver the ICE probe to the middle of the right atrium, rotate the catheter in the clockwise direction with a slight P curve to obtain the long-axis view of the interatrial septum, and along with the interatrial septum, its junction with the superior and inferior vena cava can be visualized. Further rotate the catheter in the clockwise direction with P curve to obtain the short-axis view of the atrial septum. At this point, the anterior (aortic) and posterior borders of the interatrial septum can now be visualized. Once the sheath/needle drag procedure is started, the “Tenting sign” of the puncture needle can be identified in the long-axis view of the interatrial septum. Then, rotate the ICE catheter in the counterclockwise direction until both the needle tent and the mitral annulus can be visualized to determine the optimal level of the puncture site.After successful puncture, fix the puncture needle, push the dilator sheath into the left atrium, withdraw the sheath core, and the “tram track sign” can be observed in the ultrasound image. Deliver the stiffened guidewire to the left superior pulmonary vein or place in the left atrium, preferably looped for stability and ability to track the sheath. At this point, it should be determined that the activated clotting time (ACT) is within the therapeutic range before subsequent operations. The peripheral arterial balloon dilates the interatrial septum so that the sheath can smoothly pass through the interatrial septum. Adjust the A/P knob of the ICE catheter under fluoroscopy to align the catheter with the track of the stiffened guidewire, and gently push the catheter into the left atrium. In case of resistance during the procedure, slightly adjust the R/L knob or rotate the catheter in the clockwise/counterclockwise direction and push the catheter again, or adjust the ICE catheter under left or right anterior oblique fluoroscopy.After entering the left atrium, the ICE ultrasound probe can display images of the left atrial appendage, pulmonary veins, and mitral valve. Preoperative TEE for mitral valve intervention can identify most intra-atrial appendage thrombi, but when it is difficult to differentiate intra-atrial thrombi from normal pectinate muscle tissues by TEE, intraoperative application of ICE may be considered to re-identify the presence of intra-atrial appendage thrombi , 124. ReHenning et al. first reported the application of ICE in TEER . HoweverSalem et al. first reported ICE-guided PBMV , and theTranscatheter mitral valve replacement guided by CS combined with ICE can be used to treat severe mitral incompetence caused by biological valve deterioration or prosthetic valve ring dysfunction and severe mitral annular calcification (MAC) , 133. ICDue to the distance limitation of ICE imaging, the guidewire in the left atrium should be retained after mitral valve intervention, so that ICE probe can enter the left atrium for evaluation of therapeutic effect. Although ICE has shown many advantages in mitral valve intervention, it is not recommended to completely replace TEE at present due to the lack experience in relevant application, the lack of uniform operating specifications, the lack of popularity of 3D ICE and other reasons.Experts’ recommendation: (1) ICE imaging from within the left atrium can be helpful if not critical in guiding transseptal puncture and evaluating the mitral regurgitation in the mitral valve intervention; (2) However, mitral valve intervention requires a large sheath to operate across the interatrial septum, which will affect the entry of the ICE catheter into the left atrium, and the treatment of mitral valve disorders mostly requires the guidance of 3D images, so it is currently not the main recommended method.Intracardiac echocardiography can clearly show the right ventricular outflow tract, pulmonary valve, and proximal pulmonary artery. Therefore, ICE comes with a good application prospect in the transcatheter intervention of pulmonary valve. At present, there is still little experience in the application of ICE in pulmonary valve intervention, limited to guiding transcatheter pulmonary valve replacement (TPVR) , 135. InPost-TPVR infective endocarditis (IE) is a potentially fatal complication. Previous studies showed that the incidence of post-TPVR IE and transcatheter pulmonary valve-related IE was 5.1 and 1.9%, respectively . TEE is Experts’ recommendation: (1) ICE plays a good role in the assessment of pulmonary valve intervention and complications, and is not inferior to TEE; (2) Pulmonary valve intervention and ICE share the same approach and may interfere with each other; (3) It can be recommended as an effective alternative to TTE and TEE  The image of ICE in transcatheter tricuspid valvuloplasty is non-inferior to, or potentially superior to that of TEE; (2) It is recommended that patients with throat or esophageal lesions and anesthesia contraindications should receive the guidance of ICE; (3) The guidance of 3D/4D ICE will have a great role in promoting the tricuspid valve intervention , use the antegrade approach of transseptal puncture for closure, and if the PVL is close to the septal side , use the retrograde approach for closure. For most patients, the placement of the ICE catheter in the right atrium can meet the imaging requirements of the operation, and very few patients require placement of the ICE catheter in the right ventricle or even the left atrium. For patients with failed PVL closure under ICE guidance, a repeated closure or other treatments can be considered. Although disposable catheters increase surgical costs, ICE-guided PVL closure avoids the need for anesthesiologists and reduces the time of surgery. There are currently no reports on PVL closure after ICE-guided TAVR. As most ICEs on the market can only assess regurgitation bundles by 2D and color Doppler, and due to the safety concerns associated with catheter placement, their clinical application is still greatly limited. The recent advent of real-time 3D/volumetric imaging can contribute to the further display of anatomical structures by ICE, thus improving the success rate of the operation, with good application prospects.Experts’ recommendation: (1) The application of ICE in aortic and mitral PVL is worth recommending, especially in patients intolerant to TEE examination; (2) 3D/4D ICE is more helpful in PVL intervention.In LAAC, ICE is increasingly favored by surgeons and patients for its more flexible and convenient operation, richer and more comprehensive viewing angles, improved tolerance and safety, less X-ray exposure and contrast agent dosage compared with TEE. Almost all patients can tolerate ICE-guided LAAC under local anesthesia. The specific process is as follows . It focuAs ICE avoids the adverse effects and esophageal injury due to TEE probe entering the esophagus, and is well tolerated by patients, LAAC can be completed under local anesthesia. After the ICE catheter reaches the right atrium, three conventional sites, namely the right atrial body, right ventricular outflow tract and proximal segment of the coronary sinus, are usually recommended for layer-by-layer scanning of the LA/LAA to rule out the possible presence of atrial and atrial appendage thrombi, of which the right ventricular outflow tract is the best scanning site to rule out thrombi. In LAAC, if ICE is used to guide the transseptal puncture, the method for judging the level of puncture point is as follows: Select the site where the membrane at the lower end of the interatrial septum is close to the muscle; the method for judging the anteroposterior position of puncture point: The ICE sector is in the same plane as the puncture needle tip and the left superior pulmonary vein (LSPV) ridge site. After successful puncture of the interatrial septum, under the guidance of X-ray, adjust the direction of the ICE catheter tip, and deliver the ICE catheter into the LA along the direction of the guidewire; or deliver along the location of the septal marker point in a 3D electroanatomical system.The LAA is a 3D structure in the cardiac cavity, so its dimensions should be evaluated in three dimensions. The ICE catheter delivered into the LA allows close and multi-angle scanning of the LAA, avoiding the limitations of TEE application in patients with cardiac transposition and atrial appendage variations. Multi-angle assessment is the key point for ICE-guided LAAC. Routinely long-axis layer-by-layer scanning can be performed through three anatomical positions that are orthogonal, namely X axis (left pulmonary vein), Y axis (ostium of right pulmonary vein) and Z axis   are located. Send the delivery sheath into the ostium of LAA under the guidance of the pigtail catheter (as shown in After the closure device (disk) is expanded, ICE is applied to evaluate the closure effect  layer by layer from multiple angles. After the release criteria of each closure device  are met, completely release the closure device, and then perform ICE examination again from multiple angles to evaluate the release effect of the closure device, avoid the displacement of the closure device and the effect of closure device on adjacent structures , and observe whether there is pericardial effusion. If the release criteria are not met, withdraw  the closure device, adjust the position and expand again, and re-evaluate the expanding effect.It should be noted that: (1) For AF patients who meet the indications for LAAC, after successful puncture of the interatrial septum under the guidance of ICE, it is recommended that the ICE catheter be delivered into the LA or LSPV through the transseptal puncture point and the layer-by-layer scanning through “three axes and six directions” that are orthogonal from three anatomical marks be performed to assess the shape, size, and anatomical structure of the LAA. (2) A closure device of appropriate size should be selected according to the width and available depth of the LAA opening measured by angiography and ICE. After implantation of the closure device, angiography and ICE can be carried out to confirm that there is no large amount of residual shunt around the closure device, and the final pull test can be performed to examine the anchorage of the closure device.Management of arrhythmias during pregnancy is an important challenge, and arrhythmias during pregnancy will lead to many adverse effects on the mother and fetus. There are many drawbacks associated with antiarrhythmic drugs, e.g., limited drug options, significant adverse reactions, and ineffective treatment. Despite a small radiation exposure, the conventional catheter ablation still brings risks to pregnant women and fetuses, which limits its use in special populations .In recent years, as 3D electroanatomical mapping systems and ICE technique developed, completely zero X-ray catheter ablation has become feasible and in many institutions routine, which makes catheter ablation possible for the treatment of arrhythmias during pregnancy, including conventional supraventricular tachycardia and ventricular premature beats, as well as complex arrhythmias such as atrial fibrillation and ventricular tachycardia. ICE technique combined with 3D electroanatomical mapping system allows visualization of the multi-electrode catheter, facilitates electrode placement, and makes subsequent ablation of the left heart system viable after the ICE-guided transseptal puncture is completed –155. In Intracardiac echocardiography is a viable option to guide LVAD implantation, especially when other imaging modes are not appropriate. However, to avoid serious iatrogenic complications, clinicians must understand the limitations of experience, the limitations of imaging, and the risks associated with this technique. In some special cases, X-ray, TTE, and TEE are clinically not indicated for LVAD implantation, while ICE can guide the LVAD implantation in such cases. Moreover, compared with TTE and TEE, ICE can be used in patients requiring positive pressure ventilation and intubation. However, for ICE in the chambers of the heart, soft guidewire wrapping is possible during transaortic implantation of LVAD, in which case excessive tension of the guidewire may lead to a potential complication (vascular perforation). Moreover, ICE is sometimes challenging to image the descending aorta due to its limited spatial resolution, which may bring additional risks .Intracardiac echocardiography can be used as an alternative or supplement to TEE for perioperative and postoperative management of LVADs. There are case reports where the device structure, hemodynamics and related complications after ICE-guided LVAD implantation are evaluated –159. ComIntracardiac echocardiography-guided endomyocardial biopsy (EMB) is an interesting application of ICE. ICE enables precise localization for biopsy, reduces the risks associated with operations, and reduces the need for diagnostic thoracotomy, and is more maneuverable in selected cases . With thA series of patients with right ventricular mass undergoing ICE-guided EMB demonstrated safety and efficacy . The diaElectroanatomical mapping combined with ICE-guided EMB is feasible in patients with suspected arrhythmic cardiomyopathy and ventricular arrhythmia of unknown origin. This method can significantly improve the positive rate of biopsy and reduce complications  Flow ch.Radiofrequency ablation of the interventricular septum is currently a novel surgical technique for hypertrophic obstructive cardiomyopathy, and its feasibility has been demonstrated in some studies , 163. AcIn patients with aortic valve stenosis and pulmonary arterial pressure undergoing TAVR, the use of ICE instead of right heart catheterization for pulmonary arterial pressure monitoring is safe and feasible . The pulWith the increasing use of cardiac electronic implantable devices such as pacemakers, device-related complications, such as infection, thrombosis and device/lead failure, occur frequently. Transvenous lead extraction (TLE) is an important technique for managing cardiac electronic implantable device-related infections . In TLE,As a unique imaging technique, ICE can guide TLE or be combined with new techniques intraoperatively, and it is safe and feasible –180. ICEIntracardiac echocardiography has been widely used in cardiac interventions such as radiofrequency ablation, and it is also suitable for TLE based on the said advantages. With the advancement of technique and the application of 3D ultrasound imaging, the imaging quality of ICE will be further improved in the future. Therefore, the application prospect of ICE-guided TLE is promising.Nowadays, ICE has been applied in a variety of cardiovascular interventional operations. In addition to the applications introduced above, ICE can also be used to guide the implantation of leadless pacemaker , left buCurrently, catheter with a diameter of 8F has emerged, which will expand the application of ICE, such as cardiovascular interventional procedures in children and so on. With the application of 4D ICE, the improvement of image quality and the decrease of cost, ICE will be more widely used in the future.Because there are few authoritative clinical studies related to ICE in the world, some of viewpoints in our consensus are lack of considerable clinical studies to support them, and they were based on the clinical experience of the experts or case reports, we don‘t use “I-III” and “a-c” to express evidence level but use “Recommend,” “Can be useful,” “Maybe useful,” “Not recommended,”  which isAs there are few expert consensuses related to ICE in the world, the flowcharts in  in our mSome emerging technologies (such as 4D ICE and 8F catheter) related to ICE are only mentioned in this manuscript, and their clinical applications are not elaborated too much, because they are rarely used in China. We hope that we will have the opportunity to improve the expert consensus with the application of these technologies in the future.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


Rivers are the most threatened ecosystems globally. However, much attention has been directed to natural rivers as urban streams are regarded as unnatural with poor capacity to provide ecosystem services. This study was carried out to explore the capacity of an urban Palmiet River to provide refuge for aquatic biota and the importance of solid wastes in providing habitat for macroinvertebrates in severely disturbed urban rivers. The water was found to be of good quality in the upper reach/headwaters and polluted in the industrial area. However, the river showed the ability to self-purify as the water showed some improvement as it flows downstream. Various sensitive and tolerant groups of macroinvertebrates were found along the river. Although the current condition of the river can still support biodiversity and the functioning of the river, it is unclear if the system could endure further disturbance. Some sites with good-quality water showed low or absence of sensitive organisms due to poor habitat. It is evident that macroinvertebrate assemblages are influenced by the water quality and availability of physical habitat, and solid wastes may provide additional habitat in severely disturbed streams. These findings attest that urban river functioning does not differ from that of a natural river, and it should be appreciated as it provides important ecosystem services for local communities.p < 0.05) across six sites. Good-quality water was observed at Site 6, whereas Site 5 exhibiting hypertrophic condition. Sites 4 to 1 were all eutrophic; however, nutrient levels showed to decrease from Site 4 down to Site 2 and increased again at Site 1. A similar trend was observed for habitat quality, with Site 6 showing excellent and Site 5 exhibited poor habitat. Coinciding with water and habitat quality, macroinvertebrate diversity and abundance showed significant differences across six sites. Sensitive palaemonids, notonemourids, and amphipods were only observed in the headwaters and have contributed over 50% of the variation in abundance between Site 6 and other sites. The non-metric multidimensional scaling (NMDS) plot has also shown clear discrimination  for the Average Score Per Taxon (ASPT) across the six sites. Macroinvertebrate communities have shown a clear association between water and habitat quality. These findings affirm the ecological importance of urban rivers as they provide refuge to aquatic biodiversity, with anthropogenic litter providing additional habitats for other taxa. Despite the current conditions supporting biodiversity and the functioning of the river, it is unclear if the system could endure further disturbance.Urban rivers are regarded as unnatural because they drain catchments characterized by impervious surfaces. The present study explored macroinvertebrate communities in relation to water and habitat quality along the longitudinal gradient of an urban Palmiet River in Durban, South Africa. Sampling was conducted across six sites along the river. The water quality has shown a significant variation (ANOVA,  Freshwaters are known to be biodiversity hotspots, with their species constituting about 10% of species on Earth ,3. In urAs a result, urban rivers experience an extremely high flow regime during rainy seasons, which ultimately destroy natural habitats and reduce nutrient retention ,7. High Despite urban rivers showing the ability to support aquatic biota, they still received less attention as they are regarded as a highly degraded system with poor capacity to provide ecosystem services . SimilarThe Palmiet River is a unique urban stream characterized by highly variable stretches along the longitudinal gradient. The river passes through an urbanized residential area in the upper reach, followed by an industrial area in the middle reach, then a nature reserve and another urban residential area in the lower reach ,17. Alth2 catchment comprising urban residential and industrial areas with two community nature reserves, New Germany Nature Reserve and Palmiet Nature Reserve , nitrate (NO3), ammonia (NH3), potassium (K), sulfate (SO4), and phosphate (P) were measured. The guideline suggested by DWAF [Sampling was carried out seasonally from April 2017 to February 2018 to cover different hydrologic flow regimes. However, due to urban rivers being sensitive to flash flood, we sampled four weeks after the flood during flooding seasons so that the macroinvertebrates could recover. Physical variables, i.e., temperature, pH, dissolved oxygen, salinity, and total dissolved solids, were measured in situ using Hanna multiparameter instrument (Model: HI98194). Three water samples were collected randomly in a 1 × 10 m transect using 1-L sampling bottles at each sampling site during each season. The river depth was mostly <0.5 m, so the water was sampled from approximately half the depth. Samples were kept in a cooler box filled with ice. The samples were later sent to an accredited laboratory for chemical analysis. In the laboratory, nitrite  was used to evaluate seasonal variation for water variables, habitat scores, Shannon-Weiner and Simpson’s Diversity indices, species richness, and abundance in each site and across six sites. The confidence interval for ANOVA was set at ggplot2”  was usedp < 0.05). The pairs of sites which showed significant differences were Site 1–Site 5, Site 4–Site 2, Site 5–Site 2, Site 3–Site 5, Site 6–Site 4, and Site 6–Site 5. A significant difference has been observed for temperature across the six sites  with dissolved oxygen showing no significant difference . A significantly higher level of TDS was observed at Site 5, with Site 6 showing lower TDS  (p < 0.05) (Results for water variables are reported in  < 0.05) . Coincid < 0.05) , with a  < 0.05) . Nitrite < 0.05) . Sulfate < 0.05) .p < 0.05), with only Sites 1 and 2 showing similar habitat conditions (p > 0.05). The lower habitat score was observed at Site 5 (52%) which is within the industrial area. Site 1 has shown a good quality habitat with an IHAS score of 72% being recorded. This river stretch has shown heterogeneity in vegetation biotope, from riparian to aquatic vegetation. The water depth was below 0.5 m with no anthropogenic modification on the channel. Moreover, stone biotopes exhibited a high heterogeneous habitat with high variability of stone sizes. There was a considerable length of gravel and sand habitat with dark mud characterized by dead leaves and tree logs. Site 2, which is in the Palmiet Nature Reserve, showed a similar river stretch (68%) to Site 1 with good quality for all biotopes. Habitat scores have shown a significant difference across six sites . Located in the headwaters was Site 6, which was characterized by a narrow channel and predominantly sand and mud habitat, with scatted heterogeneous vegetation habitat, roots, and tree logs. This site exhibited relatively shallow and clear water. A considerably high IHAS score was observed in this site (76%). Using a habitat quality classification protocol   (Taxa recorded in this study are presented in  < 0.05) . A notab < 0.05) . Oligoch < 0.05) . Palaemop < 0.05), with lower richness being observed in Site 5. Similarly, a significantly lower taxa diversity was observed at Site 5 compared to all other sites   . Most ta < 0.05) . Turbell < 0.05) . p < 0.001), with average distances to median being 2.82, 2.94, 2.83, 2.76, 2.14, and 3.06 for Sites 1 to 6, respectively. In contrast, significant results were observed for PERMANOVA (p < 0.001).Corroborating the species richness results was the SASS5 scores which showed clear distinction across the six sites . The mea2, NO3, NH3, and P being within US-EPA [The water quality has shown spatial variation along the longitudinal gradient of the Palmiet River. The headwaters of the river showed ideal water quality with pH, NOn US-EPA , CCME [2n US-EPA , and DWAn US-EPA  guidelinn US-EPA ,31. Siten US-EPA . It is eHowever, the water quality improves as the river flows downstream, with Site 2 showing much improved conditions. In contrast, Site 1 showed another increase in water quality variables which suggests another pollution just outside Palmiet Nature Reserve. According to Tian et al.  and ŠaulWater quality parameters have shown no seasonal variation for all sites. Urban rivers are draining impervious surfaces, hence, susceptible to flash floods . TherefoUrban streams are usually characterized by poor habitat due to constant disturbance induced by humans, resulting in channel modification and nutrient enrichment ,40. HoweMacroinvertebrates are important components of lotic aquatic ecosystems, and they showed to respond rapidly to habitat destruction, change in hydrologic regime, and water quality deterioration ,44,45. TDespite physico-chemical variables, physical habitat is known to influence macroinvertebrate assemblage . AlthougSites 1 to 3 showed no substantial difference regarding taxa diversity, richness, and abundance. These sites were characterized by almost similar habitats and water quality. The most abundant taxa in these sites were coenagrionids, naucorids, lestids, baetids, and hydropsychidae, consecutively. No association was observed between the water quality and taxa diversity across these three sites. Contrastingly, a strong association was observed between taxa diversity and habitat scores. Coenagrionids and lestids inhabit emergent vegetation, beatids occupy rocky substrates, hydropsychidae inhabit decaying tree logs, whereas naucorids are found crawling on the bottom substrate . These sDespite the spatial variation, Sites 1, 2, and 6 showed seasonal variation for total taxa abundance, whereas taxa richness showed seasonal variation for all sites. Taxa abundance was substantially lower at Sites 5 and 4. Site 5 constantly receives effluents from industries, so it is evident that change in hydrologic flow regime has not influenced pollution load in this site. A similar trend was observed at Site 4, which showed no seasonal variation. Site 4 is located just downstream of Site 5, which suggests that there was a slight water quality improvement at Site 4 and little to no seasonal variability with regard to anthropogenic litter. This supports the idea that macroinvertebrate assemblages in urban streams can be influenced by numerous factors such as flow regime, water quality, habitat availability, etc. ,51.Generally, changes in diversity and abundance of aquatic biota along the longitudinal gradient of rivers due to local environmental conditions have been widely observed ,52. The Macroinvertebrates as indicators of water quality have been used for decades, and they proved to be the most reliable pollution screening tool in both urban and natural rivers ,49,53,542, NO3, NH3, and P. Therefore, the ASPT observed in the present study has successfully shown clear discrimination across sites with different water quality. It is evident that ASPT remains a more reliable tool to evaluate environmental conditions in an aquatic ecosystem compared to community structure and abundance.Nevertheless, the scores showed to improve as the river flows downstream, with Site 3 peaking again. This site was characterized by much-improved water quality and good quality habitat compared to Sites 4 and 5. According to Khudhair et al.  habitat Urban rivers are highly appreciated by urban dwellers, and their ecological integrity is of utmost importance . This st


One of the pathological hallmarks of Alzheimer’s disease (AD) is the formation of amyloid-β plaques. Since acetylcholinesterase (AChE) promotes the formation of such plaques, the inhibition of this enzyme could slow down the progression of amyloid-β aggregation, hence being complementary to the palliative treatment of cholinergic decline. Antiaggregation assays performed for apigenin and quercetin, which are polyphenolic compounds that exhibit inhibitory properties against the formation of amyloid plaques, reveal distinct inhibitory effects of these compounds on Aβ40 aggregation in the presence and absence of AChE. Furthermore, the analysis of the amyloid fibers formed in the presence of these flavonoids suggests that the Aβ40 aggregates present different quaternary structures, viz., smaller molecular assemblies are generated. In agreement with a noncompetitive inhibition of AChE, molecular modeling studies indicate that these effects may be due to the binding of apigenin and quercetin at the peripheral binding site of AChE. Since apigenin and quercetin can also reduce the generation of reactive oxygen species, the data achieved suggest that multitarget catechol-type compounds may be used for the simultaneous treatment of various biological hallmarks of AD. Alzheimer’s disease (AD) is ranked as the third cause of death in the world after heart diseases and cancer, and is the most common cause of adult-onset dementia, affecting currently around 50 million people worldwide . This neThe multifactorial nature of AD is reflected by the distinct hypotheses that have been proposed for its etiology, e.g., the cholinergic neuron damage, the amyloid-β (Aβ) cascade, oxidative stress and inflammation, among others . The choAs a result of the complex etiology of AD, intense research is focused on the development of multitarget compounds that encode the linkage of both synthetic and naturally occurring scaffolds ,21,22. AIn this context, the present study aims at providing a comprehensive analysis of the potential AChE inhibition and Aβ antiaggregating activity of flavonoids, paying particular attention to the potential effect of AChE in promoting the formation of Aβ fibrils and how the presence of flavonoids may affect the interaction between AChE and Aβ ,16. To tElectrophorus electricus (AChE), acetylthiocholine iodide (>99.0%), 5,5′-dithiobis(2-nitrobenzoic acid) , quercetin and apigenin were obtained from Sigma-Aldrich. All solutions were prepared using native buffer .Aβ40 was purchased from Bachem . Thioflavin-T (ThT), acetylcholinesterase from Aβ40 (1 mg) was solubilized in 500 μL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) under vigorous stirring at room temperature for 1 h. The resulting solution was sonicated for 30 min and subsequently stirred at room temperature for 1 h. The solution was maintained at 4 °C for 30 min to avoid solvent evaporation during aliquot collection. To eliminate possible insoluble materials, the samples were filtered over 0.22 μm filters. Lastly, aliquots of soluble Aβ were collected, and the HFIP was evaporated under a gentle stream of dinitrogen. The samples were stored at −20 °C.ex = 445 nm; λem = 480 nm) was used to detect the formation of Aβ fibrils with an Aminco-Bowman Series 2 luminescence spectrophotometer .Aβ samples were resuspended in 50 μL of DMSO, and the monomers were solubilized through sonication for 10 min. Typically, a solution containing Aβ40 (20 µM), 50 µL of DMSO, 850 µL of native buffer and 100 µL of ThT is used to study the formation of fibrils. This solution is split into two parts; 10 µL AChE (from a stock solution of 200 μM) is added to one of them. These two solutions (V = 500 µL) are split again giving four solutions (V = 250 µL), two of them containing AChE. A 0.5 µL volume of inhibitor (10 mM) was added to one of the samples containing AChE. The samples are subsequently incubated at 37 °C for 24 h. The controls are incubated at 4 °C for 24 h. Fluorescence spectroscopy with ThT  is obtained under the boundary conditions of t = 0 and f = 0, where k = kea (a being the protein concentration). By nonlinear regression of f against t, the values of ρ and k can be determined, and from them, ke and kn, which are the elongation and nucleation kinetic rate constants, respectively  is the concentration of the flavonoid tested; qK is the quenching rate constant; τ0 is the average lifetime; and svK is the Stern–Volmer quenching constant.The fluorescence quenching was obtained by applying the Stern–Volmer equation (Equation (3)):aK is the binding constant, and n is the number of binding sites per AChE molecule. The assays were performed in triplicates, and SD values <5% were obtained in all cases.The binding constant was determined using Equation (4), which illustrates the relationship between the fluorescence quenching intensity and the concentration of the tested flavonoid.The assay based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) was carried out as described in the literature . Increas−3 mol·L−1), 16 µL of AChE (0.5 U·mL−1), 9 μL of flavonoid (at the required concentration) and 665 μL of NB were prepared. Then, 200 μL of sol A was mixed with 1, 2, 3 or 4 μL of ATC (15 × 10−3 mol·L−1) to start the reaction that was followed at 412 nm (absorbance) for 2 min, allowing to obtain the enzymatic ratio.The mechanism of AChE enzymatic inhibition was determined using Lineweaver–Burk reciprocal plots. Briefly, a solution (sol A) containing 200 μL of DTNB , 50 µL of DMSO and 950 µL of native buffer was used. This solution was split; 10 µL of AChE was added to one of the solutions. The two solutions (of 500 µL) were again split, giving four solutions of 250 µL, two of them containing AChE. A 0.5 µL volume of inhibitor (10 mM) was subsequently added to all samples. The samples containing AChE were incubated at 37 °C for 24 h and the controls at 4 °C for 24 h. The particles suspended in liquids (PSD) were measured with DLS.−1 within the range 4000–550 cm−1. All spectral data were acquired and baseline-corrected using the Omnic v9.2 software.Attenuated total reflectance–FTIR spectra of lyophilized samples were registered with an FTIR Thermo Scientific Nicolet iS5 spectrometer equipped with a diamond ATR iD7 device. Each spectrum accumulated 64 independent scans, measured at a spectra resolution of 2 cmCarbon-coated copper grids of 200 mesh were activated through glow discharge for 30 s. Immediately after, the samples were deposited onto the grids, which were thoroughly washed with Milli-Q water. Then, the grids were treated with UranyLess and left to dry in a desiccator for at least 24 h prior to visualization by TEM. The samples were visualized using a Tecnai Spirit TWIN (FEI) 120 kV TEM microscope equipped with a LaB6 emitter and a Megaview 1 k × 1 k CCD.g for 60 min, and the soluble fractions were analyzed by Mini-PROTEAN® Tris-Tricine Precast Gels (16.5% bis-acrylamide) . The gels were stained with Coomassie brilliant blue, scanned at high resolution, and the bands were quantified with GelEval (FrogDance Software v1.37).Aβ40, purchased from Bachem, was dissolved in 50 μL of DMSO at a concentration of 400 µM, and the monomers were solubilized through sonication for 15 min. Native buffer (750 µL) was added, and the sample was divided into four aliquots (190 µL). Then, 0.8 µL of each compound (apigenin or quercetin) at 6 mM in DMSO was added  or 0.8 µL of DMSO without compound. Finally, 47.5 µL of AChE (20 µM) was added to obtain a final concentration of 4 µM. The samples were incubated in a thermomixer  at 37 °C and stirred at 1400 rpm for 24 h. The negative control is left at 4 °C. The next day, 100 µL of each sample was mixed with 100 µL of Congo Red (CR) at 100 µM and incubated at room temperature for 2 h. After incubation, the samples were precipitated by centrifugation at 16,000× The binding mode of quercetin to human AChE (hAChE) was investigated by combining molecular docking, molecular dynamics (MD) simulations, which were used to refine the binding mode, and free-energy calculations, which were carried out to estimate the binding affinity.Docking was performed using three X-ray structures of hAChE  chosen as protein templates in order to consider the distinct arrangement of residues Tyr337 and Trp286 , which are located in the catalytic and peripheral pockets, respectively . Docking−1 Å−2 to preserve the binding mode of quercetin, avoiding the occurrence of artefactual changes during equilibration, at constant pressure (1 bar) conditions. Starting from the equilibrated structures, three replicas (R1-3) were run for each system. Production calculations were performed at constant volume and temperature using SHAKE  (As illustrated in avonoid] c. Both favonoid] . The datavonoid] .Electrophorus electricus enzyme (eeAChE), computational studies were performed considering the X-ray crystallographic structures of hAChE deposited in the Protein Data Bank with codes 4M0F, 6CQU and 6O4X. This choice was motivated by several reasons. First, the lack of crystallographic structures for the eeAChE enzyme, since the low resolution achieved in previous studies impeded the modeling of the details of the backbone and side chains [To explore the molecular basis of the AChE inhibition, the binding mode of quercetin to AChE was investigated by combining molecular docking and MD simulations. Although the AChE inhibitory activity was experimentally determined using the e chains . Second,e chains ,61. KeepThe docking results pointed out a preferential binding of quercetin at the peripheral site in all cases, excluding the feasibility of binding at the catalytic site. Remarkably, this agrees with the noncompetitive inhibition mechanism found for this compound (see above) and previous studies that reported that only PAS inhibitors can block the AChE-induced aggregation ,18,19.–1. The best pose was obtained in the docking to 4M0F and 6O4X , likely reflecting the formation of π–π stacking interactions between quercetin and both Trp286 and Tyr341. In contrast, an outer pose was found in the docking to 6CQU , though still retaining contact with Trp286. In line with these results, the top-ranked binding modes obtained for 4M0F, 6O4X and 6CQU were refined by means of MD simulations. The RMSD profile obtained for the protein backbone was stable along the trajectories, and only minor adjustments were observed for the residues that shape the peripheral site, as expected from the solvent exposure of this binding pocket  and 4M0F—Replica 3  are the most stable complexes, followed by 6CQU—Replica 2 , 4M0F—Replica 2  and 6O4X—Replica 3 . This reflects the potential capabilities conferred by the hydroxyl groups attached to the core of this flavonoid, suggesting that the interaction of quercetin at the peripheral site may involve more than a single binding mode.The contributions to the binding affinity determined for these poses are shown in The antioxidant properties of some antiamyloid agents may represent a beneficial factor, contributing to their global antiaggregation activities. The radical scavenging abilities of apigenin and quercetin were thus studied using DPPH. As illustrated in The results obtained in the present study reveal the effects exerted by flavonoids apigenin and quercetin on the interplay between (i) the inhibition of the enzymatic activity of AChE, (ii) the formation of Aβ40 fibrils and (iii) the AChE-promoted Aβ aggregation.0 relative to free Aβ, suggesting the occurrence of favorable interactions between these compounds and Aβ40 in the first steps of the aggregation, altering the normal fibrillation pathway. The presence of AChE leads to a notable reduction of the time required to complete the Aβ fibrillation; the analysis of the kinetics parameters reflects that the reduction of the nucleation process is counterbalanced by an increase in the elongation rate constant, leading to a decrease in the half-aggregation time. Incubation with apigenin and quercetin alters the influence exerted by AChE on Aβ40 aggregation, leading to an inhibition of 25.5 and 57.9%, respectively. Nevertheless, the extent of this effect is clearly mitigated compared with the antiaggregating activity of these compounds in the absence of AChE, as the inhibition was 66.5 and 74.9% for apigenin and quercetin, respectively.The reduction in the formation of Aβ fibrils observed in the presence of quercetin and apigenin is reflected by the increase in the nucleation rate constant and the decrease in the lag time tThe reduction of the antiamyloid activity of the flavonoids in the presence of AChE is indicative of interactions between the flavonoids and AChE. The noncompetitive mechanism observed in the enzymatic inhibition assays, together with the decrease in the fluorescence of the enzyme (presumably due to the interaction with Trp286), agrees with an interaction of apigenin and quercetin at the peripheral site of the enzyme, as also shown by molecular modeling studies, which reveal the interaction with Trp286. Noteworthy, the lower association constant determined for the binding of quercetin to AChE provides a basis to justify the larger effect observed on the kinetics of the AChE-induced aggregation.Overall, these findings suggest that a proper choice of the chemical groups that decorate the flavonoid scaffold can be exploited to finely tune the balance between the AChE inhibition, Aβ40 fibrillation, and AChE-Aβ interaction. In conjunction with the antioxidant properties (relief of oxidative stress) and the ability to reach the central nervous system , flavono


Sea-level rise projections rely on accurate predictions of ice mass loss from Antarctica. Climate change promotes greater mass loss by destabilizing ice shelves and accelerating the discharge of upstream grounded ice. Mass loss is further exacerbated by mechanisms such as the Marine Ice Sheet Instability and the Marine Ice Cliff Instability. However, the effect of basal thermal state changes of grounded ice remains largely unexplored. Here, we use numerical ice sheet modeling to investigate how warmer basal temperatures could affect the Antarctic ice sheet mass balance. We find increased mass loss in response to idealized basal thawing experiments run over 100 years. Most notably, frozen-bed patches could be tenuously sustaining the current ice configuration in parts of George V, Adélie, Enderby, and Kemp Land regions of East Antarctica. With less than 5 degrees of basal warming, these frozen patches may begin to thaw, producing new loci of mass loss. This study uses ice sheet modeling experiments to show that thawing portions of the Antarctic ice sheet bed can increase century-scale mass loss, particularly in the Wilkes and Enderby Land regions of East Antarctica. While frozen bed conditions beneath the continental interior6 inhibit basal sliding, basal temperatures at, or close to, the pressure melting point (PMP) enable the fast-flow of glaciers and ice streams near the ice-sheet margin7. In some regions, thawed-bed outlet glaciers or ice streams are interleaved by frozen-bed ice rises or ice ridges9, resulting in frozen and thawed basal regimes in close proximity to each other , self-reinforcing feedbacks22 mediated by temperature-dependent sliding23 could lead to the spontaneous onset of wet-bedded, fast flowing ice and the emergence of ice streams21. However, the effect of changes in the basal thermal state has not been constrained by observations or modeling25, so the impact on the dynamics and stability of the Antarctic Ice Sheet remains unknown.It is possible that Antarctica’s basal thermal state could evolve on centennial timescales. Considering the continuous increase in atmosphericthawable to refer to regions where the bed is frozen but within a few degrees below the PMP  to reduce the modeled basal friction coefficient to capture the effect of a frozen-to-thawed transition in enhancing the slipperiness at the bed. This method enables us to conduct a first-of-its-kind investigation of the effect of basal thawing on ice mass loss, even though it lacks thermo-frictional feedback mechanisms that are needed to fully resolve the ice sheet response to basal thaw.We use a novel approach to simulate basal thawing in thawable-bed regions. A frozen-to-thawed bed transition would, in theory, reduce basal friction and accelerate sliding30.We run four model experiments with the following thawable amplitudes within the experiment zones: For Exp. 1, we define the thawable-bed regions as the regions where basal ice temperature amplitude is −2 °C – PMP; Exp. 2 is −5 °C – PMP; Exp. 3 is −8 °C – PMP, and Exp. 4 is −12 °C – PMP 1/2). We then extract the model inferred α values32 in the experiment zone and group them into thawed, thawable, and frozen ranges based on the basal temperature output from the model initialization. For each temperature range, we present the corresponding α values as empirical Cumulative Density Functions (CDFs) , there are higher α values in frozen and thawable regions and lower α values in thawed regions  in the CDF  is run for 100 years to provide a benchmark to the other experiments. The ice sheet geometry, velocity, and grounding lines are allowed to evolve in time in all simulations.To prescribe thawing in the thawable regions, we first define basal sliding in the model set-up using a Weertman-type friction lawwed Fig. . In eachCDF Fig. . We refe34 friction law to capture drainage-related effects, (ii) modification of α by a prescribed percentage of its original value (rather than the CDF method), and (iii) extending thaw areas further into the ice sheet interior. These additional experiments are discussed in Supplementary Note To examine the robustness of the experimental set-up, we perform three sets of additional experiments: (i) replacing Eq.  with a Bα values are held constant. On a continental scale, more aggressive thawing produces more mass loss relative to the control for all basins , Marie Byrd Land (F-G), and the Bellingshausen Sea (H-H’) regions, our modeling results display little to intermediate mass loss (0.5–2.5 Gt/yr) with moderate thawable areas in Exp. 1 and Exp. 2, and up to about 4 to 5 times that for the most extreme thawing scenario represented by Exp. 4. Perhaps surprisingly, in the Amundsen Sea Embayment where recent observations already show significant mass loss (>100Gt/yr)33, our results suggest that this region is relatively insensitive to further basal thawing.In West Antarctica, the mass loss in the Siple Coast and Ellsworth Mountain basins  have the lowest relative mass loss, which is somewhat expected since the basal temperatures are well below −12 °C. Notably, our model highlights that the Enderby-Kemp and George V - Adélie Land regions (D-D’ and A’-B) of East Antarctica would likely undergo the most mass loss and be most sensitive to changes in basal thawing. These regions lose ~20 Gt/yr for Exp. 4, doubling the rate of other nearby basins. This number is also an order of magnitude higher than the observed dynamic loss for these regions over the last four decades33.In East Antarctica, along the Wilkes Land coast up to MacRobertson Land and the Dronning Maud through Shackleton-Pensacola regions  has substantial thawable areas  can be attributed to the basin-specific conditions. While the thawable extent is fairly high  and Enderby - Kemp Land (A’-B): these regions comprise ~11% of the total ice sheet area and could be susceptible to mass loss an order of magnitude higher than the observed dynamic loss over the last four decades24. Our results likely represent an upper-bound on expected mass loss, since warmer conditions are introduced abruptly and over the entire thawable regions. In reality, we would expect such changes to happen more progressively, and they may occur in only one region. Further, our experiments are built around an idealized representation of basal thawing that does not account for geometric and thermal feedbacks that could result in the bed eventually refreezing.In addition, our results highlight the need for advances in modeling the processes leading to thawing and the ice sheet response to environmental forcing47. Our work motivates the need for more model development in this area. The latter question requires an investigation of refreezing. One pathway that could lead to refreezing is a feedback between faster sliding speeds driven by thawing and the ice geometry, where increased flow speed would lead to thinning and therefore larger conductive heat loss to the surface. This process entails a rearrangement of the englacial temperature field, thus our 100-year simulations are probably barely long enough to start seeing the impact. Another pathway is that thawing may imply a large enough reduction of basal friction that frictional heating would no longer be large enough to maintain a positive melt rate, despite the faster sliding speeds. This would be instantaneously apparent in our simulations. Our simulations do not show refreezing in thawable areas, which suggests that they could remain thawed after their initial frozen-to-thawed transition  What physical processes can lead to basal thawing? (2) Under what conditions and over what timescales can a thawed bed be sustained? Modeling the physical processes capable of driving basal thawing would require a continent-scale ice sheet model with Stokes mechanics at high spatial and temporal resolution to resolve sliding onset physicsAs a first assessment of ice mass loss sensitivity to basal thawing, our work clearly suggests that there is a relationship between basal thawing and mass loss, particularly in parts of East Antarctica. The considerable mass loss that we find in response to basal thawing raises the possibility that physical processes driving basal thaw are part of a feedback that should be considered as a mechanism contributing to sea level rise. To assess this, we propose that follow-on work should investigate the spatial extent and timescales of physical processes that could potentially force and sustain thawing. Additionally, there is a need for observational constraints of the basal thermal state particularly in the George V, Adélie, Enderby and Kemp Land regions of East Antarctica where our model results show that parts of the bed are within a few degrees of thawing. These advances in observationally constraining the basal thermal state and including the physics of basal thaw in model projections of the Antarctic ice sheet’s future evolution are critical steps to understand the effects of basal thaw, a hitherto neglected process in the context of sea level rise projections.26. Ice is treated as a viscous incompressible material with strain and temperature-dependent viscosity following Glen’s Law48. For the stress balance calculation, we use a 3-D higher-order approximation to the Full Stokes equations50. The ice sheet surface is stress-free and at the ice-bed interface we apply a power-law version of the friction law51 that is implemented in ISSM in terms of the basal shear stressα is the basal friction coefficient, N is effective pressure, 51. We explore the sensitivity of the model to the choice of friction law, with a comparison between results obtained with Weertman and Budd friction reported below in Supplementary Note p = 1 and q = 0 in Eq. 55. We use surface velocity from the MEaSUREs InSAR-based Antarctic Version 2 velocity map (https://nsidc.org/data/nsidc-0484)57. The remaining surface conditions are surface temperature and surface mass balance from the 1979–2014 climatological annual mean from MAR v 3.6.458. We use ice shelf basal melt rate values from ref. For the Antarctic ice sheet geometry, we use BedMachine Antarctica v 1 (60 and the layer spacing is refined towards the bottom (extrusion exponent of 1.2) to better resolve the rapidly changing temperature structure and vertical shearing in the bottom part of the ice thickness. The 3-D mesh comprises ~800,000 prismatic elements.Our model domain covers the entire Antarctic ice sheet. We use an adaptive mesh with horizontal resolution ranging from 1 km along the coast to capture narrow fast flowing glaciers to 30 km in the continental interior to maintain reasonable computational cost. In the vertical direction, we use 5 layers with quadratic interpolation functions (P1xP2 finite element type)32 to infer the basal friction coefficient, α, on the grounded ice sheet, and similarly for depth-average ice rigidity, B, over ice shelves61. The cost function is1 is the coefficient for the term representing the absolute difference in velocity, γ2 is the coefficient for the log of the misfit, and γt is the coefficient for the regularization term and ε is the minimum velocity used to avoid singularities. We choose are γ1 = 400, γ2 = 1, and γ1 = 100, γ2 = 1 and there is no regularization term. For the first iteration, the ice rigidity temperature dependence is calculated from observed surface temperatures, which allows us to compute an initial guess for the depth-resolved ice sheet velocity before we have a model temperature field.The initial model configuration is computed via an iterative initialization procedure that seeks to compute the 3D velocity and temperature fields for a known, present-day ice sheet geometry. Initially, we treat the mechanical and thermal problems separately. First we consider the mechanical problem, where we use the same inverse method presented in ref. 53. We stabilize the heat advection solver with the anisotropic streamline upwind Petrov–Galerkin (SUPG) method which circumvents the problem of an ice sheet’s high aspect ratio by treating the horizontal and vertical directions separately in the stabilization coefficients62. For the first iteration step, we solve a diffusion only problem for the temperature field. We then successively iterate our initialization procedure for the mechanical and thermal problem: the ice velocity field is used to update the temperature field by solving the steady, 3-D advection-diffusion problem for temperature53, this updated temperature is then used to update the velocity field via the solution of the inverse mechanical problem described above. To ensure full consistency between the thermal regime and the stress balance regime we run multiple iterations of the mechanical and thermal model until the mean basal temperature varies less than 0.05% between iterations. Also, we apply 20% random noise as perturbation to the friction coefficient values on our final two iterations to ensure that the inversion optimization does not get stuck in a local minimum.For the thermal problem, we assume a thermal steady state throughout the initialization24 . To update the mesh at each time-step of the 100-year simulations, we use the arbitrary Eulerian–Lagrangian method67, where the vertical elements adjust according to the ice surface while the horizontal mesh remains fixed. We choose a fixed bimonthly time step (48 steps per year). We chose this fixed time step after testing runs with adaptive time stepping to determine the maximum time step that satisfies numerical stability criteria. The control shows a small increase in the total ice sheet volume above flotation of 0.23% over 100 years (Supplementary Fig. 68 reported a 0.14% increase in total ice sheet volume above floatation over 100 years for a continent wide control simulation using ISSM and Pattyn et al.69 reported 0.2–0.3% increase for a continent wide simulation using the f.ETISh model.For all 100-year experiments, the climate conditions and ice shelf basal melt rates are held constant, so the only perturbations come from Supplementary Information


Toxoplasma tachyzoite replication and sexual differentiation in Plasmodium. However, apicomplexan MAPK signaling is notable for the lack of the canonical kinase cascade that normally regulates the networks, and therefore must be regulated by a distinct mechanism. We highlight what few regulatory relationships have been established to date, and discuss the challenges to the field in elucidating the complete MAPK signaling networks in these parasites.Reversible phosphorylation by protein kinases is one of the core mechanisms by which biological signals are propagated and processed. Mitogen-activated protein kinases, or MAPKs, are conserved throughout eukaryotes where they regulate cell cycle, development, and stress response. Here, we review advances in our understanding of the function and biochemistry of MAPK signaling in apicomplexan parasites. As expected for well-conserved signaling modules, MAPKs have been found to have multiple essential roles regulating both  The mitogen-activated protein kinases (MAPKs), together with their regulators, comprise a core eukaryotic signaling module. The canonical MAPK module translates extracellular signals across a 3-tiered kinase cascade to provoke cellular responses , and MAPK2  from here on.Three distinct MAPKs have been identified in Apicomplexa: ERK7, MAPKL1, and MAPK2 , Table 1-1/PfMRP ,12. The  PfMAP-2 . ToxoplaTgMAPKL1  (originaTgMAPKL1 ), TgMAPKTgMAPKL1 ), and TgTgMAPKL1 ,16). TheMammalian ERK7  has been described as able to robustly autophosphorylate both the Thr/Tyr of its activation loop, and thus autoactivate . Such auCryptosporidium and in the Chromerids. It would therefore appear that each of the 3 apicomplexan MAPKs were found in the ancestral organism and that these proteins originally evolved in the context of a fully functional MAPK cascade.While it is possible that ERK7 is the ancestor of all extant apicomplexan MAPKs, such a model is not supported by phylogenetic analysis . The numWhile the apicomplexan MAPKs are divergent, phylogenetic analysis robustly supports their identification as bona fide members of the family  Fig 2)..Fig 2). P. berghei is required for its nuclear localization, due to 2 nuclear localization signals (NLSs) in the region -X-[I/L]) and are unfolded until binding [Two distinct docking sites have been identified on the MAPK kinase domain. The first, called the D-site or common docking (CD) domain, is thought to be present in all MAPKs. The D-site lies on the face opposite of the kinase active site and binds so-called “kinase-interacting motifs” or KIMs –46 Fig . Most of binding ,48. Some binding . The sec binding ,50 and t binding  Fig 3)..2-4-[I/L binding –53. EvenYersinia, use proteins with optimized docking site motifs to compete off the endogenous partners of kinases such as mammalian RSK and ERK2 [In addition to regulation by phosphorylation, MAPKs, like other kinases, may be inactivated by binding inhibitor proteins. The scaffolding protein PEA-15 sequesters metazoan ERK2 in the cytoplasm . PEA-15 and ERK2 ,55, therToxoplasma. We demonstrated that Toxoplasma apical cap protein 9 (AC9) binds tightly to the ERK7 kinase domain, and is required for its recruitment to the parasite apical cap, and thus for its essential function [D approximately 50 nM), the distribution of the binding energy over such a large surface leads to an interaction that is relatively dynamic. This led to a model whereby AC9 acts both to concentrate TgERK7 at its site of action and to block phosphorylation of nonspecific substrates. AC9 can be effectively competed off by substrates containing motifs that recognize the D-site with only moderate affinity (1 to 10 μM) [Such a mechanism of regulation is likely especially important for kinases such as ERK7 that are autoactivating. Indeed, we have found that that an inhibitory scaffold is essential to ERK7 function in function . AC9 intfunction   . In facto 10 μM) . Furthero 10 μM)  and faciToxoplasma MAPKs show strong cell cycle dependence of their transcript levels . While TgERK7 protein is found throughout the cell cycle [Another major mechanism of kinase regulation is, of course, protein homeostasis. Each of the 3 upathDB; ,59). Allll cycle , TgMAPKLll cycle . TgMAPK2Toxoplasma and Plasmodium, we will focus on the current understanding of MAPK function in those organisms.Recent work has defined functions for each of the apicomplexan MAPKs . As the Toxoplasma tachyzoites, and use of a temperature-sensitive allele demonstrated that its loss-of-function results in over-duplication of centrosomes [Toxoplasma and related organisms [TgMAPKL1 localizes to the outer core of the centrosome of dividing trosomes . This lerganisms ; MAPKL1 Notably, MAPKL1 has been demonstrated to be susceptible to inhibition by at least 2 available compounds that are marketed as selective. The ALK4,5,7 inhibitor SB505124 was demonstrated to block parasite replication both by inhibiting MAPKL1 and by altering host HIF1α signaling . MAPKL1 lasmodium, MAPK2 was found to be essential for male gametogenesis and ex-flagellation, a process by which the male gametocytes undergo 3 rounds of replication leading to the production of 4 flagellated gametes [P. berghei asexual stages, early attempts to knock out the kinase in P. falciparum in the blood stages [Plasmodium MAPK2 is primarily nuclear-localized in gametocytes [In P gametes ,64. Whild stages  led to td stages ,64. Notaetocytes , expressetocytes .Plasmodium and conservation throughout Alveolates, MAPK2 is essential in the asexual tachyzoite stage of Toxoplasma [Somewhat surprisingly, given its function in xoplasma , though xoplasma . This apxoplasma . Thus, TToxoplasma both manifest in opposing phenotypes associated with centrosome duplication. While the loss of TgMAPKL1 results in over-duplication of the centrosome, loss of TgMAPK2 blocks duplication. Interestingly, while TgMAPKL1 is tightly associated with the centrosome [Intriguingly, MAPKL1 and MAPK2 loss-of-function in ntrosome , TgMAPK2ntrosome . Thus, Tntrosome ,67, whicntrosome ,69. One Toxoplasma ERK7, which is essential to the tachyzoite lytic cycle [Toxoplasma tachyzoites replicated normally within a vacuole, but were immotile and therefore incapable of egress and invasion of new host cells [Compared to metazoa, where it is one of the most poorly understood MAPKs, in Apicomplexa, the best understood MAPK pathway is that of ic cycle . TgERK7 ic cycle , though st cells . In parast cells . Recent We recently completed an interactome study of TgERK7 . In addiToxoplasma, the Plasmodium falciparum ERK7 ortholog (PfMAP-1) is apparently dispensable both for the blood stage and mosquito stages of the parasite’s life cycle [Plasmodium ERK7 localization is dynamic as parasites develop: P. berghei ERK7 was found to be enriched in the parasite nuclei in early liver stage schizonts. In the cytomere stage, PbERK7 was instead concentrated to comma- and ring-shaped structures that no longer co-localized with the nucleus, while in merozoites ERK7 displayed uniform cytosolic localization [In stark contrast to findings in fe cycle . What thlization . It is uMuch of the fungal and metazoan MAPK signaling networks were elucidated by a combination of genetic screens, early systems biology methods , and painstaking biochemistry. Identifying the full complement of activators, negative regulators, scaffolds, and downstream substrates of the apicomplexan MAPKs will take similar efforts. Importantly, the depth of understanding of human MAPK signaling has led to >10 FDA-approved small molecules to date  and a plethora of tool compounds that target these pathways. Kinase signaling therefore represents an, as yet, untapped bounty of targets to treat apicomplexan infections. Of course, modern proteomics and bioinformatics methods can greatly accelerate discovery. However, to gain the depth of knowledge required to unravel the complexity of parasite kinase signaling will require the same kind of careful, hypothesis-driven cell biological, and biochemical work that enabled our current understanding in well-studied models.