Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, 'calcium sparks', determine the contraction of the myocardium. We adapted a correlative super-resolution microscopy protocol to correlate sub-plasmalemmal spontaneous calcium sparks in rat right ventricular myocytes with the local nanoscale RyR2 positions. This revealed a steep relationship between the integral of a calcium spark and the sum of the local RyR2s. Segmentation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR2 clusters. In myocytes taken from failing right ventricles, RyR2 clusters themselves showed a dissipated morphology and fragmented  clusters. They also featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyR2s. While fragmented  RyR2 clusters were rarely observed directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight, non-random clustering of the local RyR2 in both healthy and failing ventricles. However, the two-dimensional spatial patterns of spontaneous Ca2+ sparks in the sub-plasmalemmal cytoplasmic space of rat right ventricular myocytes were observable with TIRF microscopy. In TIRF image frames, sparks appear as a transient (approx. 30–100 ms) and localized event of fluorescence with a full-width-at-half-maximum (FWHM) of 1–6 µm (a). The current benchmark for mapping RyR2 channels in cardiomyocytes is DNA-PAINT. With approximately 10 nm of resolution, DNA-PAINT represents a significant (approx. 25-fold) improvement in optical resolution over standard diffraction-limited methods such as TIRF (b). With DNA-PAINT, it is now possible to resolve individual RyR2s, observed as punctate labelling densities, that make up each RyR2 cluster (c). In this study, we sought to selectively map the two-dimensional spatial properties of sub-plasmalemmal sparks against the local, two-dimensional organization of RyR2 located within peripheral couplons  in healthy and failing RV myocytes using TIRF microscopy.Caf 1–6 µm a. The cu as TIRF b. With D cluster c. In thie). In this protocol, RV cardiomyocytes were isolated freshly from rat hearts and transferred to a glass dish with a gridded bottom, allowing the cells to adhere to the grid. Cytoplasmic fluorescent [Ca2+]i indicator Fluo-4 AM was loaded into cells and de-esterified before cells were placed within the TIRF field. Sub-plasmalemmal Ca2+ sparks were recorded from cells at specific grid coordinates onto two-dimensional time series. Cells were then fixed in situ and labelled with anti-RyR2 antibodies towards DNA-PAINT imaging and then returned to the TIRF microscope, tracked based on their grid coordinates and DNA-PAINT series recorded. Post-hoc analysis included rescaling and a semi-automated alignment of a time-averaged facsimile of the Ca2+ time series against the DNA-PAINT image, primarily using the unique outline of the myocyte to overlay the Ca2+ sparks with the underlying RyR2 map . The coordinates of both the Ca2+ sparks and the individual RyR2 puncta were detected and subsequently registered against each other . This allowed the RyR2 channels and clusters within the ‘spark footprint’  to be counted and sampled on a spark-by-spark basis (h). The percentage histograms of the total number of RyR2 puncta  and the number of unique RyR2 clusters detected with this method in healthy RV myocytes  within the footprint of each spark demonstrate that spontaneous Ca2+ sparks can arise from broadly varying ensembles of RyR2 channels (mean of apporx. 18.1 RyR2 per spark) and unique clusters .To this end, we developed a novel experimental pipeline detailed recently  figure e. In thiRyR2 map f,g. The rk basis h. The pe. 2.22+ sparks in the failing RV, we examined myocytes acutely isolated from the RVs of rats administered with monocrotaline (MCT-RV) and age and sex-matched controls (Ctrl-RV). In the correlative, spark-by-spark analyses in MCT-RV, we observed a∼30% increase in the total number of RyR2 (mean of approx. 23.5 RyR2 per spark) and a approximately 60% increase in the number of segmented clusters  compared to Ctrl-RV. This was consistent with the likely recruitment of a greater number of RyR2 in the genesis of a spontaneous Ca2+ spark. Spontaneous sparks recorded using TIRF in MCT-RV cells also featured, on average, approximately 38% greater ‘spark mass’  compared to Ctrl-RV . While the FWHM was unchanged between MCT-RV and Ctrl-RV, the spark frequency was >90% higher in MCT-RV .To study the structure–function relation of Caa,b; see arrowheads in panel b). With the use of correlative sparks recorded over approximately 20 s, we generated ‘heat maps’ of the local nanoscale RyR2 morphology  to indicate the average estimated spark mass of the locally recorded spontaneous Ca2+ sparks in Ctrl-RV and MCT-RV cells . In the healthy control, the colour-gradations in the spark mass maps were relatively evenly distributed across clusters of similar size while smaller, fragmented clusters were sparse. In the failing cells, we observed a large majority of the fragmented  RyR2 clusters in darker colours, suggesting that they alone are unlikely to produce sparks with greater spark mass.Close examination of DNA-PAINT images of sub-plasmalemmal RyR2 from correlative experiments showed a noticeably more dissipated morphology in MCT-RV cells compared to Ctrl-RV . In Ctrl-RV, sparks with spark mass <1.0 correlated with almost exclusively with regions consisting of no more than 25 RyR2 puncta. By contrast, sparks with spark mass <1.0 were correlated with a broader range of RyR2 counts . This heterogeneity in MCT-RV is further illustrated in the frequency histograms of the total RyR2 count and the number of unique RyR2 clusters underneath each spark , represented by the wider distributions of MCT-RV compared to Ctrl-RV. Further, the direct ratio between the recorded spark mass and the locally counted RyR2 showed a lower mean and median for MCT-RV  compared to Ctrl-RV . The increased heterogeneity in MCT-RV was represented by an∼2.2-fold higher standard deviation compared to the control .The scattergrams between the spark mass of each Ca Ctrl-RV e,f. In C. 2.32+ sparks recorded using TIRF over a window of time onto the two-dimensional super-resolution image of the intricate rows of sub-plasmalemmal RyR2 clusters (a). We investigated the non-random distribution of the spark centroids visually observed across the field of view by performing a two-dimensional quadtree segmentation of domains with higher density of aggregation of spark centroids over a 15 s time window . This segmentation revealed two-dimensional, sub-plasmalemmal regions, typically a few hundred nanometres in width, throughout a cell's footprint which we classified as ‘recurring spark sites'. Figure 3b,c illustrate the overlays between these recurring spark sites and the super-resolution RyR2 images in Ctrl-RV and MCT-RV respectively . Strikingly, we observe that the recurring spark sites rarely overlap with larger RyR2 clusters directly. Instead, they appear to extend between adjacent clusters in both Ctrl-RV and MCT-RV. There was also no observable, consistent co-location of the fragmented RyR2 with the recurring spark sites in MCT-RV. The occupancy of the recurring spark sites between RyR2 clusters, often within hundreds of nanometres, resembled the shared ‘RyR2 super cluster domains’ proposed previously as a structural correlate of clusters that are likely to co-activate i that can simultaneously model the cellular and molecular basis of cardiomyopathy. To our knowledge, only DNA-PAINT o, this fraction was reported to be small i with a Ca2+ ionophore due to the requirement of preserving the cellular structure for DNA-PAINT. As a standardization, all experiments were performed under identical excitation intensities and camera settings which allowed use to quantitatively compare the intensity information Ca2+ images between cells and samples.The key determinants of the latter are the resolution and the SNR of the averaged Ca. 3.22+ sparks i can vary by 2–3 orders of magnitude.The scattergrams between spark mass and local RyR2 counts e,f demonannulled f. In fac. 3.32+ handling proteins over nanometre/micron length-scales  need to be considered with the 30% increase in the RyR2 ensemble observed underneath Ca2+ sparks. While the individual cluster size of RyR2 is reduced in MCT-RV as a consequence of the cluster fragmentation or fraying [2+ channel expression [2+]i in MCT-RV myocytes, offering greater driving force during Ca2+ release. The super-resolution heatmaps encoding spark mass do not however reveal a clear correlation between fragmented RyR2 and regions with higher average spark mass. This observation may however be different if Ca2+ leak could be mapped with a more sensitive approach than diffraction-limited TIRF imaging with Fluo-4 Ca2+ indicator.Increasing structural heterogeneity, including local t-tubule remodelling –47, locah-scales , are nowh-scales  and the h-scales  and BIN-h-scales . In this ATPase) ,50 along fraying ,32, the . 4. 4.12+ imaging and DNA-PAINT were acquired using the open-sources Python Microscopy Environment [All experiments were performed on a Nikon TE2000  modified to enable TIRF dual-colour imaging with 488 nm and 671 nm excitations on an approximately 15 µm × 15 µm illumination field (as detailed previously ). The fuironment  (PyME) s. 4.2Experiments were performed according to the UK Animals (Scientific Procedures) Act of 1986 and with UK Home Office approval (license number 70/8399) and local ethical approval. As a model of right ventricular (RV) failure, adult male Wistar Crl rats aged approximately 5 weeks were given an intraperitoneal injection of crotaline  to induce pulmonary arterial hypertension. Age and sex-matched controls were given an equivalent bolus of saline. At 3–4 weeks, the MCT treated animals were monitored for signs of RV failure were euthanized along with age-matched Ctrl animals. Hearts were dissected acutely and right ventricular myocytes enzymatically isolated following Langendorff perfusion. For a detailed account of the animal model and cell isolation, see the electronic supplementary material, methods section.. 4.32+ indicator (ThermoFisher Scientific) and immersed in a Tyrode's solution containing 5 mM CaCl2 at pH 7.4 and adhered to a gridded imaging dish with a #1.5H glass coverslip bottom  for 90 mins before being immersed in fresh Tyrode's solution for Ca2+ spark imaging. The dishes were clamped securely onto the stage of the TIRF microscope system such that the grid was aligned with the straight edges of the camera's field of view, under brightfield illumination. Cells forming a substantial footprint were illuminated with a 488 nm laser. The local changes in the Fluo-4 fluorescence were recorded at a frame rate of 10 Hz with either Tetraspeck microspheres (ThermoFisher Scientific) attached to the imaging grid or the outline of the cell's contact patch with the grid included within the imaging frame. See detailed protocol in the electronic supplementary material, methods section.RV myocytes were loaded with Fluo-4 AM fluorescent Ca. 4.42+ imaging, cells were fixed in situ with 2% paraformaldehyde  for 10 mins at room temperature (RT). Samples were washed and subjected to immunofluorescence labelling with a monoclonal mouse anti-RyR2 IgG  primary antibody and an anti-mouse IgG secondary antibody (Jackson Immunoresearch) conjugated to a DNA-PAINT P1 ‘docking’ strand  was app. 4.5et al. [2+ imaging were used to return the field of view to the corresponding cells imaged previously. For exciting the Atto655 on the imager strands marking the RyR2 targets at the very edge of the cell, the 671 nm laser was focused at a supra-critical angle onto the field of view to achieve evanescent TIRF illumination. Time series of the DNA-PAINT events consisting of 20 000–50 000 frames were acquired at 100 ms/frame integration time. The analysis included the detection of single molecule events and least-squares fitting of a two-dimensional Gaussian to localize their sub-pixel scale centroid. The event positions were then rendered into a 16-bit greyscale TIFF image with a pixel scaling of 5 nm per pixel using an algorithm based on Delaunay triangularization [Following immunolabelling, samples were washed 3 times in ‘Buffer C’ formulated by Jungmann et al. , and therization .. 4.62+ spark data to the DNA-PAINT data and the localization of Ca2+ sparks has been published elsewhere [The full image analysis protocol consisting of the registration of the Calsewhere .. 4.6.12+ spark localization tool ImageJ plugin software: ‘xySpark’ [2+ sparks. Output of this analysis was a list of x, y and t coordinates of each spark along with their full-width at half maximum (FWHM) estimated by the Gaussian fit, coefficient of determination R2, amplitude estimated as F/F0, where F0 was the estimate of the baseline level of the Ca2+ indicator fluorescence in a local cytoplasmic region and F was the fluorescence intensity value at the peak of the spark. Only sparks with 1.0 µm ≤ FWHM ≤ 6.0 µm (to select for in-focus sparks) and an R2 value ≤ 0.5  were filtered and retained for further analysis, as established previously by Hurley et al. [The CaxySpark’  was usedy et al. .. 4.6.2The punctate RyR2 labelling densities in the rendered images were detected using a centroid detection algorithm available through PyME and describe previously . The lis. 4.6.32+ images and align the coordinates of the Ca2+ sparks to the DNA-PAINT RyR2 image, as described recently [2+ time series were averaged to produce a low-noise, diffraction-limited image of the cell. This image was taken as a reference image of the cell's relative position against the Tetraspeck microsphere fiduciary markers and the cell boundaries. The code upscaled the low-noise Ca2+ image to match the pixel scaling of the DNA-PAINT image. It then required the user to manually align the Ca2+ image against the DNA-PAINT image, using either the cell outlines and/or the Tetraspeck fiduciary markers as guides. The x and y shift coordinates used for this alignment were then used as a starting point to perform an automated fine-alignment through cross-correlation of the images. The shift coordinates determined through the fine alignments were then applied to the Ca2+ spark coordinates align them against the maps of RyR2 puncta. See Hurley et al. [We used an image alignment pipeline code to upscale the Carecently . Brieflyy et al.  for deta. 4.6.4F/F0), FWHM3 and conversion factor 1.206, and was one of the default output parameters of the xySpark software [e,f) represent individual sparks . Biological replicates  are also stated in Figure legends .Data comparing RyR2 counts and spark properties did not meet the criteria for parametric tests; therefore the comparisons between MCT-RV and Ctrl-RV represent Mann–Whitney


Moreover, we have used molecular docking and molecular dynamics simulations to understand the structural determinants of RoT that explain its ability to inhibit AQP3. Our results show that RoT blocks AQP3-glycerol permeation by establishing strong and stable interactions at the extracellular region of AQP3 pores interacting with residues essential for glycerol permeation. Altogether, our multidisciplinary approach unveiled RoT as an anticancer drug against tumors where AQP3 is highly expressed providing new information to aquaporin research that may boost future drug design.The natural polyphenolic compound Rottlerin (RoT) showed anticancer properties in a variety of human cancers through the inhibition of several target molecules implicated in tumorigenesis, revealing its potential as an anticancer agent. Aquaporins (AQPs) are found overexpressed in different types of cancers and have recently emerged as promising pharmacological targets. Increasing evidence suggests that the water/glycerol channel aquaporin-3 (AQP3) plays a key role in cancer and metastasis. Here, we report the ability of RoT to inhibit human AQP3 activity with an IC Mallotus philippinensis species are known to contain different natural compounds, mainly phenols, that were reported with a vast array of biological activities such as antimicrobial, antioxidant, antiviral, cytotoxicity, antioxidant, anti-inflammatory, immunoregulatory and anticancer activities, and have been widely used as traditional medicine in several countries [Mallotus philippinensis, rottlerin (RoT) has demonstrated antitumor properties in a variety of human cancers, suggesting its potential as a therapeutic agent for cancer treatments [2O2) [ountries . Among teatments . RoT waseatments ,4 althoueatments , antiangeatments , anti-ineatments , anti-aleatments , anti-mieatments , anti-fueatments , anti-paeatments  and oxideatments . Likewiseatments . Until neatments . While teatments ,14,15. Featments . Studieseatments , in parteatments , has neveatments ,19. In heatments ,21. Accoeatments ,23. A fots [2O2) .2O2, modulating oxidative stress and triggering signaling cascades responsible for cell proliferation and migration [AQPs were reported to be aberrantly expressed in different human tumors being implicated in cancer cell proliferation and migration, angiogenesis, and metastasis ,25,26, sigration ,31. Amonigration ,33,34. Higration ,36.Despite all the studies characterizing RoT antioxidant and anticancer properties, its effect as a modulator of AQPs has never been explored. This prompted us to investigate the possible effect of RoT  on AQP1 f) and glycerol (Pgly) permeability coefficients were calculated from the rate of cell volume change after the osmotic shock. RoT effect was obtained by treating cells with RoT (25 µM for 30 min at RT).To assess the inhibitory effect of RoT on AQP1 and AQP3 activity, we first screened the effect of RoT on water and glycerol permeability in hRBCs, that endogenously express AQP1 (water channel ) and AQPgly), possibly via the glycerol channel AQP3, and a 52% inhibition of water transport (Pf) that can be mediated by both AQP1 and AQP3. To determine RoT potency, the IC50 values for the inhibition of water and glycerol transport were obtained through permeability assays ranging RoT concentrations from 0.1 to 150 µM. The obtained dose–response curves demonstrate a strong glycerol inhibitory potency (Pgly inhibition IC50 = 6.7 ± 2.97 µM) and a significant inhibition of water transport (Pf inhibition IC50 = 22.8 ± 5.82 µM) (As shown in 5.82 µM) D,E.Saccharomyces cerevisiae model, previously optimized by our lab, and used for heterologous aquaporin functional studies [Since the observed inhibition of hRBCs water transport may be due to the blockage of AQP1 or AQP3 or both, we used the yeast  studies . Yeast cf for hAQP1-expressing yeast cells (3.002 ± 0.428 × 10−3 cm s−1) was seven-fold higher than the empty vector (0.439 ± 0.089 × 10−3 cm s−1) and, as expected, hAQP1 did not permeate glycerol, showing Pgly values similar to the cells harboring the empty vector   D. The mu cm s−1) D, confirSince no experimentally determined structure of AQP3 is currently available at the Protein Data Bank , we haveA high structural convergence of the AQP3 protein is observed after 200 ns of simulation in all replicate simulations , a valueSince our goal focused on the identification of representative conformations of the AQP3 protein monomers in a binding “ready” state, we have identified two distances between four loops found at the extracellular surface of the protein that were used as references. These two distances are clearly identified in As shown in To evaluate the dynamics of interaction established between RoT and the AQP3 binding site, and the convergency of the docking poses previously reported, we have performed MD simulations of the 15 different complexes previously determined. These interaction configurations were re-integrated in the configurations of the AQP3 homotetrameric system previously simulated and used as the starting points for the MD simulations of RoT and AQP3 complexes. As shown in 50 for the inhibition of water permeability is higher than for glycerol.As can be seen in RoT is currently considered a multitarget drug that can be cytotoxic for different cancer cell types having a successful anticancer action ,45,46. DAquaporins have been associated with cancer invasion, and metastasis  and, in 50 in the micromolar range. The higher IC50 value for water permeability compared to glycerol could account for the lower reduction in water permeability induced by RoT. These results were further validated in yeast cells transformed with human AQP1 and AQP3 and revealed the selectivity of the compound for AQP3 with no measurable effect on AQP1. The confirmed AQP3 expression and localization at the cell plasma membrane previous to functional assays, indicates that RoT has a direct effect on AQP3 channel activity with no interference in upstream protein translocation mediated by Protein Kinase C.This study reveals the inhibitory effect of RoT on AQP3, impairing water and glycerol membrane permeability of hRBCs, with an ICThe possible mechanism of RoT inhibition was investigated by computational approaches, using a generated 3D model of the protein that was further validated, followed by molecular docking studies to predict the most plausible binding configurations. According to our computational predictions, RoT establishes strong and consistent interactions with several residues found at the extracellular surface region of AQP3, acting as a stereochemical cap that blocks glycerol from accessing the pores of the protein. Both the benzenotriol and chromenol chemical groups confer the required stability of RoT to act as a lid, which is further boosted by the high flexible phenylpropyl that established strong interactions with several hydrophobic residues around the aromatic filter at the pore entrance facing the extracellular medium. The strong binding of RoT at the extracellular surface, close to the selectivity filter, explains its high affinity that prevents glycerol from permeating through the AQP3 protein, although some permeation of water molecules may still occur.In conclusion, we were able to demonstrate that RoT affects AQP3 function, and this effect can also explain the vast anticancer activity reported for this polyphenol compound. Our calculations allowed a thorough structural characterization of the interaction between RoT and AQP3, evidencing vital structural groups essential for binding. Further studies to validate the herein proposed binding mechanism are paramount for the future development of RoT derivatives with improved affinity to AQP3, potentiating its anticancer therapeutical application. Overall, our study adds new knowledge to the aquaporin research field and highlights RoT as a promising natural lead compound for the development of anticancer therapeutics for tumors where AQP3 is highly expressed.The compound Rottlerin (RoT) was obtained from a commercial database  and was solubilized in DMSO to 10 mM final concentration (stock solution).Venous blood samples were obtained from healthy human volunteers following a protocol approved by the Ethics Committee of the Faculty of Pharmacy of the University of Lisbon (Instituto Português de Sangue Protocol SN-22/05/2007). Informed written consent was obtained from all participants.g, 10 min) at room temperature (RT) and washed with PBS . Cells diluted in PBS (0.5% suspension) were immediately used in the assays.Venous blood samples were collected from anonymous human donors in citrate anticoagulant . Red blood cells were isolated by centrifugation . Escherichia coli transformants were maintained and grown in Luria–Bertani broth (LB) with ampicillin (100 µg mL−1) at 37 °C [Saccharomyces cerevisiae 10560-6B MATα leu2::hisG trp1::hisG his3::hisG ura352 aqy1D::KanMX aqy2D::KanMX (YSH1770) was used as a host strain for heterologous expression of human AQP1 and AQP3. To transform the yeast cells, the complementary DNA (cDNA) from human aquaporins (AQP1 and AQP3) was amplified by PCR from the pWPi-DEST plasmid  and C-tew/v) glucose and supplemented with adequate requirements for prototrophic growth [600 nm ≈ 1, harvested by centrifugation , washed three times and re-suspended in ice-cold sorbitol  up to a concentration of 0.33 g (wet weight) mL−1 and kept on ice for at least 90 min. Prior to osmotic challenges, cells were preloaded with the nonfluorescent precursor 5-(and6)-carboxyfluorescein diacetate  that is cleaved intracellularly by nonspecific esterases to generate the membrane-impermeable fluorescent form carboxyfluorescein (CF) known to remain in the cytoplasm [Yeast cultures were grown at 27 °C with orbital shaking in yeast nitrogen base (YNB) without amino acids (DIFCO), with 2%  measurements, a hyperosmotic shock solution containing a non-permeable solute was used  producing an osmotic gradient that results in water efflux and consequent cell shrinkage. For glycerol permeability (Pgly) measurements, a hyperosmotic shock solution containing a permeable solute was used  creating an inwardly directed glycerol gradient. After the first fast cell shrinkage due to water efflux, glycerol influx in response to its chemical gradient is followed by water with subsequent cell re-swelling.Experiments were performed on a HI-TECH Scientific PQ/SF-53 stopped-flow apparatus, with a 2-ms dead time and temperature-controlled, interfaced with an IBM PC/AT compatible 80,386 microcomputer. After challenging cell suspensions with an equal volume of shock solution at 23 °C, the time course of volume change was measured by following the 90° scattered light intensity at 400 nm or fluorescence intensity (excitation 470 nm and emission 530 nm). For each experimental condition, 5–7 replicates were analyzed. Baselines were acquired using the respective incubation buffers as isotonic shock solutions. For osmotic water permeability (Pf was estimated by Pf = k (Vo/A)(1/Vw (osmout)∞) (cm s−1), where Vw is the molar volume of water, Vo/A is the initial cell volume to area ratio, (osmout)∞ is the final medium osmolarity after the applied osmotic gradient and k is the single exponential time constant fitted to the light scattering or fluorescence signal of hRBCs shrinkage [gly was calculated by Pgly = k (Vo/A) (cm s−1), where Vo/A is the initial cell volume-to-area ratio, and k is the single exponential time constant fitted to the light scattering signal of glycerol influx in erythrocytes [For hRBCs, Phrinkage . Pgly wahrocytes .For yeast cells, the fluorescent glycerol traces obtained were corrected by subtracting the baseline slope that reflects the bleaching of the fluorophore. Optimization of yeast permeability values was accomplished by numerical integrations using a mathematical model implemented in the Berkeley Madonna software (version 10.2.8) as described .50) was calculated by nonlinear regression of dose–response curves (GraphPad Prism software) using the following equation: y = 100/(1 + 10((LogIC50-X)×HillSlope)); where HillSlope describes the steepness of the family of curves.To assess the effect of RoT, cells were incubated with different concentrations of the compound for 30 min at RT before stopped-flow experiments. The inhibitor concentration that induces 50% inhibition .All the experiments were performed in three biological and at least three technical triplicates. Data are presented as mean ± standard deviation (SD). Statistical analysis between groups was performed by two-way ANOVA and non-parametric Mann–Whitney test. A Since no experimental structure for AQP3 homotetramer is currently resolved, we have used a comparative modeling approach to generate a 3D model of this target protein. To identify a template structure that we could use as a reference to generate a 3D model of AQP3, we ran a BLAST  on UniPrTo integrate the chosen model on a pure POPC bilayer membrane, we used the InflateGRO2 method ,59. ThisMD simulations of the AQP3 protein complexed with RoT were also performed to evaluate the dynamics of the interaction of the compound with AQP3. The topology of RoT was obtained from the Automated Topology Builder and Repository ,74, and All molecular docking calculations were performed using AutoDockFR . This pr


Parametric mapping sequences in cardiovascular magnetic resonance (CMR) allow for non-invasive myocardial tissue characterization. However quantitative myocardial mapping is still limited by the need for local reference values. Confounders, such as field strength, vendors and sequences, make intersite comparisons challenging. This exploratory study aims to assess whether multi-site studies that control confounding factors provide first insights whether parametric mapping values are within pre-defined tolerance ranges across scanners and sites.A cohort of 20 healthy travelling volunteers was prospectively scanned at three sites with a 3 T scanner from the same vendor using the same scanning protocol and acquisition scheme. A Modified Look-Locker inversion recovery sequence (MOLLI) for T1 and a fast low-angle shot sequence (FLASH) for T2 were used. At one site a scan-rescan was performed to assess the intra-scanner reproducibility. All acquired T1- and T2-mappings were analyzed in a core laboratory using the same post-processing approach and software.p = 0.067; for global T2 in ms: site I: 40 ± 2 [39–41]; site II: 40 ± 1 [39–41]; site III 39 ± 2 [39–41]; p = 0.543).After exclusion of one volunteer due to an accidentally diagnosed cardiac disease, T1- and T2-maps of 19 volunteers showed no significant differences between the 3 T sites The online version contains supplementary material available at 10.1186/s12968-023-00954-9. Non-invasive quantitative myocardial tissue characterization based on parametric T1- and T2-mapping has entered clinical application several years ago and has proceeded to be one of the main techniques applied in contemporary cardiovascular magnetic resonance (CMR) imaging . Based oA cohort of N = 20 healthy volunteers was prospectively recruited and screened for eligibility. Participants were eligible to participate in the study in the absence of any cardiovascular, pulmonary, endocrine, or renal conditions and gave written informed consent and were over the age of 18 years. Exclusion criteria were any contraindications for CMR, pregnancy, breastfeeding or claustrophobia. Ethical approval was obtained from the local ethics committee of Charité Medical University Berlin . The study was retrospectively registered (ISRCTN14627679).FIT), sites II and III with 3 T scanners (PrismaFIT) . Scanner at site II is used for clinical scans, the other scan sites are research scanners only. Sites II and III were trained before the start of the study. During the study scans were monitored by vendor provided software . At scanner sites I and III an 18-channel body surface coil was used and at scanner site II a 30-channel body surface coil was used : site I with 3 T scanner  sequences. A full short axis (SAX) stack covering the entire left ventricle (LV) and four long axes including a 4-chamber view (cv), 3-cv and 2-cv as well as a right ventricular (RV) view, were acquired. Parametric T1 -and T2-mapping was acquired in the three SAX slices: basal, midventricular and apical, based on the 3-out-of-5 method . T1-mappAll images were analyzed with dedicated commercially available software  by two readers with 8 (R.F.T.) and 3 years (J.G.) experience in CMR. Cardiac function and mass were evaluated as per current recommendations in SAX cine images with delineation of papillary muscles , 24. MapGiven normal distribution, as assessed by the Shapiro Wilk test, all continuous variables are given as mean ± standard deviation (SD) with the 95%-confidence interval and, where appropriate, as percentage. All categorical variables are given as total and percent. Comparisons for global, basal, midventricular, apical as well as basal and midventricular septal means for T1 and T2 were carried out for sites I, II and III. In addition, AHA segment means were compared. As a global test a repeated measures ANOVA was applied. In cases in which the significance level set at < 0.05 was reached, pairwise comparisons were carried out between the sites with a Bonferroni correction. The 95%-confidence intervals of the differences between sites I, II and III were calculated and compared to previously defined 95%-tolerance intervals (for T1 mapping ± 24.5 ms and for T2 mapping ± 3.2 ms) to assess for equivalence . Equival2; body surface area 1.9 ± 0.2 m2) all underwent scans at site II and 18 at sites I and III. LV and RV function parameters, provided as total and indexed concerning body surface area and height where appropriate, were as follows: LV end-diastolic volume: 181 ± 42 ml/96 ± 17 ml/m2/100 ± 20 ml/m; LV stroke volume: 113 ± 27 ml/60 ± 17 ml/m2; LV ejection fraction: 62 ± 3%; RV end-diastolic volume: 204 ± 51 ml/108 ± 21 ml/m2; RV stroke volume: 107 ± 27 ml/56 ± 11 ml/m2; RV ejection fraction: 52 ± 4%. Further general characteristics for the travelling volunteers at each site are presented in Table Of the 20 volunteers one had to be excluded from the final analysis as a cardiovascular comorbidity was diagnosed shortly after the scans. From the remaining 19 volunteers  age 26.1 ± 6 years; weight 70.2 ± 11.4 kg; height 1.8 ± 0.1 m; body mass index 21.7 ± 2.4 kg/mThe highest rate of analyzable segments for T1-mapping was noted at site II with a total of 266/304 segments (88%) used for final analysis after exclusion of artefacts. Sites I (245/288 (85%) showed a similar percentage, with the lowest one at site III (231/288 (80%). Higher overall rates of analyzable segments were found for T2-mapping .Of the total of 138 encountered artefacts, 124 (90%) were susceptibility artefacts. The remaining artefacts were due to mispositioning of a slice  12/138 (9%) and 2 [2/138 (1%)] due to motion artefact/cardiac ghosting. Most artefacts were located in the midventricular slices [58/138 (42%)] followed by basal [40/138 (29%)] and apical locations [40/138 (29%)]. Segmental analysis revealed the majority of artefacts being in AHA segment 11 [29/138 (21%)] trailed by segment 16 [24/138 (17%)] and 5 [15/138 (11%)]. Other segments in order: 1 [2/138 (1%)], 2 [2/138 (1%)], 3 (5/138 (4%)], 4 [12/138 (9%)], 6 [4/138 (3%)], 7 [0/138 (0%)], 8 [0/138 (0%)], 9 [4/138 (3%)], 10 [14/138 (10%)], 12 [11/138 (8%)], 13 [2/138 (1%)], 14 [1/138 (1%)], 15 [13/138 (9%)]. The overall rates of slice and segmental involvement were similar if divided by sites. At site I 17/43 (40%) artefacts were in midventricular segments with segment 11 most often involved [8/43 (19%)]. At this site only susceptibility artefacts were encountered. Site II had 17/38 (45%) artefacts in midventricular slices with segment 11 most commonly involved [11/38 (29%)]. Majority [30/38 (79%)] were susceptibility artefacts. Remaining artefacts were due to mispositioned slice [6/38 (16%)] and 2/38 (5%) due to motion artefacts. Overall site III had the most artefacts with 24/57 (42%) in the midventricular slice with segment 11 being most commonly affected [13/57 (23%)]. Susceptibility artefacts accounted for most artefacts [51/57 (89%)] with the other 6 due to a misplaced slice [6/57 (11%)].p = 0.067; for global T2 in ms site I: 40 ± 2 [39–41]; site II: 40 ± 1 [39–41]; site III 39 ± 2 [39–41]]; p = 0.543)   should still be compared to the SD of this slice (± 34 ms for site I and ± 40 ms for site III). This illustrates the clinically neglectable relevance of this finding. However, controllable and non-controllable confounders which may have let to the differences between sites I/II and III, should briefly be touched upon. These include scanner version, bore size, surface coils, intrinsic physical tissue factors as well as operator experience. The scanner version of the same field strength seems to impact the results to a lesser extent as evidenced by the small differences between sites I and II, which run different scanner versions in comparison to sites II and III, which use the same scanner version. The same holds true for the bore size, which might have an effect on the results. The influence of coils as another potential confounder is difficult to assess as the sites I and III in comparison to site II used different coils however site I and III used a different scanner. One important confounder to note, however, might be the operator experience during image acquisition. This issue is unfortunately not only relevant to mapping but encompasses other aspects of CMR such as function, late gadolinium enhancement and flow assessment , 45. We With the question of interest of which of the confounding factors provided the intersite differences, this remains ultimately not answerable. More prospective data is needed to provide further insights into these findings. An introduction of prospective quality assurance as known from industry could be helpful in imaging labs as well. Nevertheless, in a retrospective setting the causes for unwanted deviations cannot be identified with certainty .A limitation of the study is the relatively small number of healthy volunteers enrolled. Neither age nor BMI were representative. The effort of transporting and locally coordinating the scans, however, was challenging as every volunteer had to be scanned within a reasonable time period. No baseline data was available, therefore no statistical estimate of sample size could be made. The differences between site III and sites I and II are currently not fully explainable by this exploratory study, potentially signifying its relevance within a larger sample size. This warrants further studies including more diverse travelling volunteers. In addition to a small and young healthy cohort, no patients with cardiac disorders were included therefore limiting statements towards detection of abnormal values at the included sites.When confounders, such as field strength, intra-vendor diversity, acquisition schemes and post-processing analysis are controlled for, parametric mapping results are comparable between sites in multicenter studies.Additional file 1. Intra- and inter-reader comparisons.


Atopy varies in people of different ages owing to different physical conditions and exposure to allergens. We aimed to cluster ages based on atopic severity using K‐means cluster analysis and identify atopic incidence, severity, as well as the association among peripheral eosinophils, IgE and sensitisation.n = 7654) with allergic symptoms and undergoing allergen‐specific IgE tests were included from 2013 to 2017. Age, sex, specific‐IgE, peripheral eosinophil counts and total‐IgE were collected.Consecutive patients (Five age categories were identified: 1–17, 18–36, 37–52, 53–69 and 70–100 years. The incidences of atopy and poly‐sensitisation decreased with increasing age. Similar trend was observed for aeroallergens, egg and milk but not for peanuts, soy or seafood. Dust mites remain the crucial factor bothering patients with allergic symptoms, especially for children and adolescents. In patients aged <52 years, sensitisation to aeroallergens was more prevalent than food. In group 37–52 years, incidence of females' atopy was higher than that of males. The overlap of atopy, high eosinophils, and high total‐IgE was found in only 19.18% of patients. The trend of allergen‐test positivity is not parallel to total IgE and peripheral eosinophil counts.Age‐grouping based on cluster analysis helps to find the changes in atopic status and distribution of sensitised allergens with age. Allergen tests are still necessary in the clinical diagnosis and treatment. An innovative exploration of the influence of age and allergens on total‐IgE and eosinophil counts is helpful for the development of bio‐targeted precision therapy.ChiCTR2300067700. Data from a random young population showed that approximately 50% of atopic patients, defined as a personal and/or familial tendency to produce IgE antibodies in response to ordinary exposure to allergens, have symptoms referable to atopy, including asthma, rhinitis and dermatitis.The incidence of atopy in random populations, defined as the presence of positive(s) on prick skin testing with a small battery of common relevant allergens, ranges from 30% to 50%.In primary hospitals, total IgE (t‐IgE) and blood eosinophils are commonly used as biomarkers for atopy or allergy. However, their predictive value for atopy requires further exploration. Therefore, a single‐centre retrospective study was performed in consecutive patients who visited the Shanghai General Hospital and their serum allergen specific IgE (s‐IgE) was assessed from January 2013 to December 2017. The aims of this study were to (1) cluster ages based on atopic severity with the K‐means cluster analysis and (2) identify the atopic severities based on the age categories, t‐IgE and peripheral eosinophils in those patients to investigate a possible relationship between these values and atopy.22.1A single‐centre, retrospective study was performed on consecutive patients who visited the Shanghai General Hospital for allergic symptoms and underwent serum allergen S‐IgE tests from January 2013 to December 2017. Patient charts were retrospectively reviewed.2.2The age, sex, date of hospital visit and serum allergen s‐IgE test results were collected and analysed. T‐IgE and peripheral eosinophil values were also collected, if available.The ethics committee of Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, approved the protocol and a waiver of informed consent was issued for our study (number: 2017KY159).2.3Artemisia argyri, dust mites, house dust, cat, dog, Blattella germanica, Penicillium notatum/branch spore would/Aspergillus fumigatus/alternaria, Humulus) and seven food allergens . Atopic severity was stratified into seven grades and assigned numbers 0–6 as the sensitive index (SI) for 0–0.35, 0.35–0.69, 0.70–3.49, 3.5–17.49, 17.5–49.99, 50–99.99 and ≥100 kU/L, respectively; of those, 17 allergens were reviewed. A positive test result was defined as s‐IgE ≥0.35 kU/L. Atopy, categorised as yes or no, was defined as at least one positive test for s‐IgE antibodies. Patients with positive anti‐CCD s‐IgE results were excluded from our analysis.S‐IgE against 17 allergen extracts and Cross‐reactive carbohydrate determinants were measured using the EUROLINE Atopy (China7) (IgE) Assay Kit, a semi‐quantitative immunoblot method. The 17 allergens could be divided into 10 aeroallergens  and scikit‐learn 0.24  were used for clustering.The analysis was conducted as follows: Step 1: k  data objects were randomly extracted and the centroids of each cluster were computed. Step 2: All data are classified into k clusters by minimising the distance from the centroids of each cluster. Step 3: The centroid of each cluster is recalculated based on the clusters reassigned in Step 2. Steps 2 and 3 are repeated until the data belonging to each cluster converge. Python Anaconda . Multiple comparisons were made using Dunn's test. Differences between aeroallergens and food allergens were compared using the Mann‐Whitney test. The threshold for statistical significance for all analyses was set at 33.1A total of 7654 patients (mean ± SD age: 42.33 ± 20.85 years) were included in this study; 3598 (47.01%) were male. The number of people who tested positive for serum s‐IgE was stable between the years . The incidence of allergies in each month was roughly stable across years Figure .T‐IgE was measured in 1863 patients and peripheral eosinophil counts were measured in 5066 patients.B. germanica and the most common food allergens were peanuts, cod/lobsters/scallops and crabs.Atopy was confirmed in 4060 (53.04%) patients Table . Each paWe investigated the variations in age among patients sensitised to different allergens Table . PatientVisual exploration of the heatmap data Figure  identifi3.2We performed the K‐means cluster analysis with five, six, seven and eight centres. The results of five cluster centres classified all patients into four age categories: 1–20, 19–42, 42–62 and 63–100 years , 18–36 (with a centroid of 28), 37–52 (with a centroid of 45), 53–69 (with a centroid of 60) and 70–100 (with a centroid of 78) years.3.3B. germanica and mould combination; sensitisation to them had the highest incidence and was the most severe in the age group of 1–17 years . The highest peripheral eosinophil count was found in patients allergic to humulus (0.27 [0.11–0.49] × 109/mL) and the lowest value was found in those allergic to soy (0.16 [0.10–0.33] × 109/mL).Peripheral and t‐IgE and eosinophil counts varied in patients allergic to different allergens Table . Patient3.5A total of 1371 patients provided the results of all three tests ; investigation (lead); methodology ; writing – original draft (lead). Jie Fang: Methodology (lead). Yong Ji: Writing – original draft . Yingying Zhang: Investigation . Xin Zhou: Conceptualization ; validation . Junfeng Yin: Formal analysis (supporting); methodology . Min Zhang: Conceptualization (lead); methodology (supporting); writing – original draft (supporting), Wuping Bao: Conceptualization (lead); investigation (lead); methodology (lead); writing – original draft (supporting).The authors declare that they have no relevant conflicts of interest.Supporting Information S1Click here for additional data file.


Reduced cortical inhibition by somatostatin-expressing (SST) interneurons has been strongly associated with treatment-resistant depression. However, due to technical limitations it is impossible to establish experimentally in humans whether the effects of reduced SST interneuron inhibition on microcircuit activity have signatures detectable in clinically-relevant brain signals such as electroencephalography (EEG). To overcome these limitations, we simulated resting-state activity and EEG using detailed models of human cortical microcircuits with normal  or reduced SST interneuron inhibition (depression), and found that depression microcircuits exhibited increased theta, alpha and low beta power (4–16 Hz). The changes in depression involved a combination of an aperiodic broadband and periodic theta components. We then demonstrated the specificity of the EEG signatures of reduced SST interneuron inhibition by showing they were distinct from those corresponding to reduced parvalbumin-expressing (PV) interneuron inhibition. Our study thus links SST interneuron inhibition level to distinct features in EEG simulated from detailed human microcircuits, which can serve to better identify mechanistic subtypes of depression using EEG, and non-invasively monitor modulation of cortical inhibition. Reduced somatostatin-expressing interneuron (SST) inhibition has been implicated in depression. However, it is impossible to establish experimentally in humans how these inhibitory changes are reflected in clinically relevant brain signals . We performed detailed simulations of human neuronal network activity and EEG signals in health and depression. We found that reduced SST inhibition led to significant changes in EEG, which accounts for changes seen in depression. Our study thus provides biomarkers that link inhibition level of a key interneuron type to measurable signatures in EEG. These biomarkers can be used to identify subtypes of depression and non-invasively monitor cortical inhibition modulation. Major depressive disorder (depression) is a leading cause of disability worldwide  and invoα5-GABAA) receptors targeted by SST interneurons resulted in pro-cognitive and anxiolytic effects [There is growing evidence that reduced cortical inhibition plays a mechanistic role in depression  and trea effects ,21. In c effects ,23. Othe effects –26.The effects of reduced SST inhibition on circuit activity could have signatures detectible in EEG, due to this cell type’s principal role in modulating input to pyramidal (Pyr) neurons in layer 2/3, which is closest to surface EEG electrodes, as well as layer 5/6 Pyr neurons whose dendrites reach the superficial layers. SST interneurons provide synaptic and tonic inhibition onto the apical dendrites of Pyr neurons ,28, and in vivo, thus meriting the use of computational models. Previous computational studies have identified inter-laminar mechanisms underlying evoked related potentials during stimulus response using simplified neuron morphologies and connectivity [Previous studies have linked neuronal spiking to extracellular signals, although mostly using animal models and local field potential (LFP) –41. Humaectivity ,45. The ectivity ,46,47 prectivity , which cectivity ,49.In this study, we identified EEG biomarkers of microcircuit effects due to reduced SST interneuron inhibition, as estimated from gene expression changes in depression ,33. UsinWe used our previous detailed models of human cortical L2/3 microcircuits  Fig 1A)Fig 1A) an = 60 randomized microcircuits, Baseline microcircuit activity was oscillatory, marked by synchronous spiking events  and corrn = 60 randomized microcircuits, p < 0.001, d = 1.67, p < 0.001, d = 2.92), and 30% decrease in offset . Together, these changes led to a 41% increase in aperiodic broadband power (5–30 Hz) in depression compared to healthy microcircuits . Detected peaks above the aperiodic component, which corresponded to periodic components, were clustered into canonical frequency bands based on center frequency. Depression microcircuits showed a 40% increase in peak periodic power within theta band , 30% increase in alpha bandwidth , 33% increase in peak periodic power within low-beta band , and 49% increase in low-beta bandwidth , but no significant change in alpha peak power compared to healthy microcircuits (p = 0.1), or center frequency for any frequency bands (p = 0.48). Thus, the absolute power increase involved a combination of an aperiodic broadband component, and periodic theta and low-beta components.We compared simulated EEG in healthy versus depression microcircuits using our previous depression models, in which SST synaptic and tonic inhibition were reduced (p = 0.3), whereas PSDs from microcircuits with 20–60% reduced SST interneuron inhibition had significantly increased power . The separability between the two types of reduced inhibition based on the raw PSD stemmed primarily from changes in decomposed aperiodic components , whereas microcircuits with reduced PV inhibition showed an increase in periodic alpha power . However, this distinction was lost at higher (60%) levels of reduction . For both moderate and high levels of reduction, the estimated bandwidth of the periodic low-beta component increased only in the case of microcircuits with reduced SST interneuron inhibition , but this parameter was less reliable since it was sensitive to the fit of the primary (larger) alpha component [To better establish the specificity of EEG biomarkers due to reduced SST interneuron inhibition, we simulated different levels of SST and PV interneuron inhibition reduction (20–60%) and compared their effects. Spectral profiles of reduced SST interneuron inhibition were distinct from reduced PV interneuron inhibition . Absolutmponents  rather tmponents . For modomponent . Periodip = 0.03, d = 0.6; 40% inhibition reduction: 7% increase, p < 0.001, d = 1.0: 60% inhibition reduction; 13% increase, p < 0.001, d = 1.8) and no significant change in offset. Contrarily, microcircuits with reduced SST interneuron inhibition showed a decrease in exponent  and no significant change in offset. The aperiodic changes resulted in microcircuits with reduced PV interneuron inhibition having a decrease in broadband power , whereas microcircuits with reduced SST interneuron inhibition having an increase in broadband power . Thus, reduced SST interneuron inhibition affected the aperiodic measures of the PSD in a manner that was distinct from reduced PV interneuron inhibition.In contrast, we found that the aperiodic component largely separated the two conditions . Microcip = 0.008, d = 0.6; 40% reduction: 0.93 ± 0.04 Hz, p < 0.001, d = 1.7; 60% reduction: 1.12 ± 0.06 Hz, p < 0.001, d = 4), but to a lesser extent than reduced SST interneuron inhibition . Thus, reduced SST interneuron inhibition affected baseline microcircuit activity to a greater extent than reduced PV interneuron inhibition.Microcircuits with reduced PV interneuron inhibition significantly increased baseline Pyr firing rate at each step , with a peak preference of 90° for all interneurons, and peak preferences of 0° and 90° for Pyr neurons. Depression microcircuits followed a similar phase preference , but there was a small decrease in preference selectivity, as quantified by population spike concentrations about the preferred phase for all cell types except SST interneurons .We identified relationships between neuronal spiking and EEG spectral changes in depression microcircuit models by determining the EEG phase preference of the neuronal populations. The EEG time series were bandpass filtered from 4–16 Hz since the absolute PSDs and the periodic component of the PSDs showed highest power in these frequencies . We compp < 0.001 for all). Thus, the EEG biomarkers corresponding to microcircuit changes in depression were mostly localized spatially.Lastly, we examined the spatial distribution of scalp-measured EEG signals generated by the microcircuit simulations using a realistic head model. Picking an example region that is also relevant to depression, we placed the simulated microcircuit dipoles into L2/3 of the dorsolateral PFC (dlPFC), at a location underneath the 10–20 system electrode F3 , and useIn this study, we identified the signatures of reduced cortical inhibition in depression on EEG signals of resting-state activity using detailed models of human cortical microcircuits. Depression microcircuits with reduced SST interneuron inhibition showed an increase in absolute theta, alpha and low beta power, which involved a broadband increase together with a periodic increase in theta and low-beta activity. These EEG signatures of reduced SST interneuron inhibition were distinct from those corresponding to reduced PV interneuron inhibition, and thus provide a measure of estimating the level of cell-specific inhibition from EEG. These signatures may thus be used as biomarkers to stratify depression subtypes corresponding to altered inhibition, which are particularly associated with treatment-resistant depression . By simuWe examined changes in EEG PSD in terms of traditionally reported absolute power within canonical frequency bands  and in tIncreased aperiodic broadband power and decreased aperiodic slope in our depression microcircuits are consistent with the effects of increased baseline activity and excitation-inhibition ratio, respectively ,73,74. IReduced SST and PV circuits both increased baseline activity but to a different degree, and had distinct EEG signatures due to their specific spatial innervation on Pyr neurons ,73,75 anReduced PV interneuron inhibition had opposite broadband effects on the PSD from SST interneuron inhibition, since decreased inhibitory currents at basal dendrites would negate apical sources and decrease the amplitude of EEG activity . The decin vivo, the simulated EEG signal magnitude can also be related to that of the recorded EEG by scaling the signal by 10,000–100,000, to account for the difference between the number of neurons in our models (1000) compared to the millions of neurons that generate the recorded EEG at a given electrode [Our biophysical models of human cortical microcircuits reproduce the two most ubiquitous features of human resting state EEG: a prominent low-frequency (4–12 Hz) peak in power and 1/f aperiodic trend ,79. In electrode ,45. Altelectrode .Linking altered mechanisms at the microcircuit scale to EEG biomarkers is currently impossible to establish experimentally in humans, thereby meriting the use of data-driven detailed computational studies. Our previous work discusses modelling considerations in detail . BrieflyWe simulated human EEG using available detailed models of human cortical L2/3 microcircuits to serve as canonical cortical microcircuit models. These layers are closest to the electrode and thus are important contributors to the EEG signal, along with L5/6 . Previouin silico simulations to improve treatment monitoring. Finally, our models and methodology may further serve to identify EEG biomarkers of altered cellular and circuit mechanisms in other neurological diseases, such as epilepsy and schizophrenia.Using detailed multi-scale models of human cortical microcircuits, we were able to characterize the EEG and spike correlates of reduced SST interneuron inhibition in depression. Previous modeling studies have shown that reduced SST interneuron inhibition in depression impairs stimulus processing due to increased baseline activity and noise . Our mod3 volume  [We used our previous models of human cortical L2/3 microcircuits , consistelow pia ). All moelow pia . Brieflyelow pia ,99 and Relow pia ,101. The.8.0.119  and LFPyon 2.0.2 . The mulon 2.0.2 ,104, andon 2.0.2 ,105,106.on 2.0.2 . The mod -80 mV) ,107,108. -80 mV) , with ot -80 mV) .in vivo [We simulated eyes-closed resting-state by injecting the microcircuit models with background excitatory input representing cortical and thalamic drive, as used previously . The bacin vivo ,110,111.We used previous models of depression microcircuits , with 40Reduced PV interneuron inhibition was modeled by iteratively reducing synaptic and tonic inhibition from PV interneurons onto all neurons by 20%, 40%, and 60%.LFPy [t from compartment n at position rn. This was done for each x, y, z component of the dipole moment, for each timestep, at each segment.We simulated layer-averaged dipole moments together with neuronal activity using LFPy . The fulLFPy  as well LFPy . Brieflymm, 80 mm, 85 mm, and 90 mm, respectively. The conductivity for each sphere was 0.47 S/m, 1.71 S/m, 0.02 S/m, and 0.41 S/m, respectively [μm) oriented normal to the surface of the scalp. The extracellular potential measured from an EEG electrode on the skin placed directly above the circuit was calculated using the following equations:r is the distance of the electrode from the center of the brain sphere, and θ is the angular separation between the dipole and the electrode. The first equation describes the potential in the innermost sphere , ϕ1, and the second describes the potential in the other 3 concentric shells , ϕs. The potential was measured at the extracellular electrode, ϕs, using a current dipole moment of magnitude p at a radial location rz. Since shell conductivity, σs, and radius, rs, affect the signal propagation, Pn(cosθ) is the n Legendre Polynomial. Full methodology for computing Corresponding EEG timeseries were generated from dipole moment timeseries using a four-sphere volume conductor model that assumes homogeneous, isotropic, and linear (frequency independent) conductivity for each medium. A four-sphere head model accurately captures EEG for sources directly under the scalp electrode ,78,113. ectively . A fixed al 2017 , with a EEG timeseries were bandpass filtered into their canonical frequency bands. The envelope for each band was calculated using the real component of the Hilbert transform from SciPy version 1.4.1 . High poWe decomposed EEG PSDs into periodic and aperiodic (broadband) components using the standard method described in the FOOOF package, version 1.0.0 . The apecos EEG cycle start (peak) and trough corresponding to 180°. For each of the four neuron populations , spike times were aggregated into a population spike vector and converted to corresponding phase of EEG. Spike phases were binned into 10° bins and the counts were summated over all circuits in each condition. To compare spike preference between conditions, bin counts were normalized by the maximal count.We calculated the instantaneous phase of EEG using the ByCycle python library version 1.0.0 . BrieflyLFPy and used in the four-sphere head model were imported into MNE version 0.24.0 [Whereas a four-sphere head model is suitable for simulating EEG from microcircuit sources directly under the scalp electrode, head shape affects EEG signal spread and thus the contribution of signals from more distant sources to the electrode . To studn 0.24.0  and downn 0.24.0  normal tn 0.24.0 , and shon 0.24.0 . We solvd to show effect size. We used Raileigh’s test of non-uniformity to determine if populations had a non-uniform phase preference, and kurtosis to quantify variance around the preferred mean angle.We used two-sample t-test to determine statistical significance where appropriate. We calculated Cohen’s


Given constrained funding for HIV, achieving global goals on VMMC scale-up requires that providers improve service delivery operations and use labor and capital inputs as efficiently as possible to produce as many quality VMMCs as feasible. The Voluntary Medical Male Circumcision Site Capacity and Productivity Assessment Tool (SCPT) is an electronic visual management tool developed to help VMMC service providers to understand and improve their site’s performance. The SCPT allows VMMC providers to: 1) track the most important human resources and capital inputs to VMMC service delivery, 2) strategically plan site capacity and targets, and 3) monitor key site-level VMMC service delivery performance indicators. To illustrate a real-world application of the SCPT, we present selected data from two provinces in Mozambique—Manica and Tete, where the SCPT was piloted We looked at the data prior to the introduction of SCPT (October 2014 to August 2016), and during the period when the tool began to be utilized (September 2016 to September 2017). The tool was implemented as part of a broader VMMC site optimization strategy that VMMC implementers in Mozambique put in place to maximize programmatic impact. Routine program data for Manica and Tete from October 2014 to September 2017 showcase the turnaround of the VMMC program that accompanied the implementation of the SCPT together with the other components of the VMMC site optimizatio strategy. From October 2016, there was a dramatic increase in the number of VMMCs performed. The number of fixed service delivery sites providing VMMC services was expanded, and each fixed site extended service delivery by performing VMMCs in outreach sites. Alignment between site targets and the number of VMMCs performed improved from October 2016. Utilization rates stabilized between October 2016 and September 2017, with VMMCs performed closely tracking VMMC site capacity in most sites. The SCPT is designed to address the need for site level data for programmatic decision-making during site planning, implementation, monitoring and evaluation. Deployment of the SCPT can help VMMC providers monitor the performance of VMMC service delivery sites and improve their performance. We recommend use of the customized version of this tool and model to the need of other programs. Voluntary medical male circumcision (VMMC) is an effective –3 and coThe current and future scale-up of VMMC and other HIV interventions must be considered in the context of HIV financing trends. Following more than a decade of significant increases, overall development assistance for HIV in low- and middle-income countries plateaued and has been declining since 2013 . Though Visual management has been used to improve the performance of production units in health care and other sectors . Visual The Voluntary Medical Male Circumcision Site Capacity and Productivity Assessment Tool (SCPT) is an electronic visual management tool developed to help VMMC providers at site level understand and improve their performance. The tool was initially developed in Microsoft Excel and then converted into a cloud application. The SCPT draws on elements of Lean. The Excel version of the SCPT was developed by USAID Project SOAR, and piloted in Manica and Tete provinces in Mozambique from September 2016 to September 2017. The web-based version of the tool was developed through the USAID supported project Supporting Operational AIDS Research (SOAR). The SCPT is a user-friendly tool capturing and displaying visual information that allows VMMC providers to: 1) track the most important labor and capital inputs to VMMC service delivery, 2) strategically plan site capacity and targets, and 3) monitor key site-level VMMC service delivery performance indicators. To our knowledge, the SCPT is the only tool that allows VMMC implementers to visually monitor inputs and outputs and assess site-level capacity and productivity. It is important to underscore that the primary focus and functionality of the SCPT is at the site level. The data the tool produces is not intended for regional or national aggregation. This article describes the online version of the SCPT. To illustrate a real-world application of the SCPT, we present examples of data from two provinces in Mozambique—Manica and Tete. We present selected data for Manica and Tete from October 2014 to September 2017 to highlight the substantial increases in VMMC coverage made over that period, and describe the role played by the SCPT in the broader VMMC site optimization strategy put in place in Mozambique from September 2016. In fact, VMMC procedures were decentralized and performed from outreach sites and /or referral to fixed sites. That is, while the number of fixed service delivery sites providing VMMC services in Manica and Tete was expanded, and each fixed site extended service delivery by performing VMMCs in outreach sites.The VMMC program in Mozambique was implemented since 2010 in the provinces with high HIV Prevalence and low male circumcision prevalence. Manica and Tete were not the only Provinces implementing VMMC. Therefore, one cannot define a non-intervention comparison because the improvement plan was designed to improve the program in Manica and Tete and not to compare Manica and Tete program performance with that of other Provinces. By 2015, the VMMC program in these two provinces were the less performing with less than 15% achievement vs target. When the SCPT was introduced as part of a comprehensive improvement strategy in 2016, by middle of 2017, the VMMC program in Manica and Tete has become the most performing program in Mozambique. It is important to highlight that the program was implemented in most of these Provinces by the same Implementing partner. However, it is not possible to attribute the site improvement and performance to one single tool or interventions because the VMMC program benefitted from a baseline assessment, the continuous quality improvement to determine performance against every single standard.This study did not entail research involving individual human participants and/or the collection of medical data for individual participants. Furthermore, interviews of patients were not conducted as part of this research. To run the SCPT requires only routine voluntary medical male circumcision program data. All program data were obtained in aggregate and made available to the research team with no names or other personal identifiers for individual patients. Therefore, it was determined by Project SOAR, Population Council and Avenir Health that ethical approval was not necessary.http://www.vmmcipt.org//, a VMMC data for decision-making toolkit portal managed by Avenir Health. Once the SCPT is accessed, resources including a detailed user’s guide and data management guidelines can be found. We describe below the current version of the SCPT and specify any important differences that exist between the online and Excel versions of the tool.The SCPT is a digital visual management tool for VMMC service providers initially developed in Microsoft Excel. To take advantage of multi-user capabilities, the SCPT was converted into a cloud application hosted on Microsoft Azure. The system architecture was built with Embarcadero Delphi backed with Excel/CSV for data storage. The front end was developed with JavaScript/React using Material-UI. The current version of the SCPT can be accessed here: The intention of the SCPT is to capture and visually present all relevant and available data in one place to enhance the ability of site managers to strategically plan VMMC service delivery. The SCPT allows users to display all the data entered in the combinations he or she chooses. SCPT users can elect to display site level data or aggregated data for a health zone, province, or region. Users can choose the time frame and format (graphs or tables) in which the data is to be displayed. SCPT users can also choose the time interval for the data display—month, quarter, half-year, or year. Besides displaying the data entered in various ways, the tool can also calculate many permutations of the information entered. For example, while users enter the number of VMMCs performed per month, the tool can generate the average number of VMMCs performed daily in a given time period calculated as the number of VMMCs performed divided by the number of days of operation during the time period of interest. In addition to facilitating the collation and reorganization of information, the SCPT also calculates several indicators using the information entered by users. These include: current site capacity, optimum site capacity, additional staff needed for optimum site operation, utilization rate, and productivity gain/loss. We describe each of these below.Current and optimum site capacity are based on existing international guidelines on optimized client flow and quality VMMC service delivery –33, reseThe utilization rate is the ratio of VMMCs performed to current site capacity. If a site performs 15 VMMCs per day and its current daily capacity is 15, then its daily utilization rate would be 15/15, or 1. If a site conducts 10 VMMCs per day and its current daily capacity is 15, its utilization rate would be 10/15, or 0.67 meaning that the site is under-used. On the other hand, if a site conducts 19 circumcisions per day and its current daily capacity is 15, its utilization rate would be 19/15, or 1.27, meaning that the site is over-used (perhaps a site requires overtime or needs to employ temporary workers to meet the demand). Both under-used and over-used sites are cause for concern. Under-utilized sites are inefficient—additional VMMCs could be performed given the staff and surgical beds available. Over-utilized sites may be stretching their resources too thinly or may be cutting operational corners to meet excess demand or overly ambitious service delivery targets. Monitoring of service quality is critical in all sites, but over-used sites should be monitored especially closely to ensure that efficiency is accompanied by service quality.The SCPT also provides program and site managers with indications of productivity gains and losses by presenting the difference between the number of VMMCs performed and the number of VMMCs the facility could have performed if operating at its optimum capacity. For example, if the number of daily VMMCs performed is 10 and the optimum daily site capacity is 10, productivity would be 10–10 = 0 or, if 10 circumcisions are performed a day and the optimum daily site capacity is 15, the productivity loss would be 10–15 = -5. On the other hand, if the number of daily VMMCs performed is 18 and the optimum daily site capacity is 15, the productivity gain would be 18–15 = 3. Both productivity losses and gains need to be explored. Productivity losses are driven by mismatches between the staff allocated to a site and the number of VMMC surgical beds in the facility. Productivity gains are driven by over-utilization in sites where current capacity has been optimized.As a case study of a real-world application of the SCPT, we present below examples of data from Manica and Tete provinces in Mozambique—where the SCPT was piloted from September 2016 to September 2017. We present selected data from October 2014 to September 2017 to showcase the improvements in VMMC service delivery the program made over the period.Over the three-year period from October 2014 to September 2017, the total number of VMMCs performed increased from 1,259 to 12,029 in Manica and from 1,024 to 15,943 in Tete . This isSeveral logistic factors including the service delivery traking using the SCPT contributed to this increase in numbers of VMMCs performed over time in Manica and Tete. The number of fixed service delivery sites providing VMMC services was expanded over time with CS Mutarara beginning to perform VMMCs in October 2016 . In addiAssessment of service delivery site indicators over time also shows that several additional site-level implementation changes accompanied the important increase in VMMCs performed in Manica and Tete between October 2014 and September 2017. Alignment between VMMC site targets and the number of VMMCs performed improved, with the number of VMMCs performed much more likely to equal or surpass VMMC site targets at the end of the period . In addiThe utilization rates—the ratio of VMMCs performed to current VMMC site capacity—in service delivery sites providing VMMC services in Manica and Tete were low in October 2014 . There wProductivity gains/losses—the number of VMMCs performed minus the number of VMMCs the site could have performed at optimum capacity—in VMMC service delivery sites in Manica and Tete varied across sites and by quarter between October 2014 and September 2017, but they were usually negative, meaning they did not perform the number of circumcisions possible based on the capacity of the site . CompariThe VMMC cascade includes the number of people reached by VMMC demand creation services, the number of people seeking VMMC services, and the number of VMMCs performed. To achieve the ambitious global goals for the scale-up of VMMC scale-up and other HIV interventions in the context of constrained resources, all providers must improve service delivery operations and use labor and capital inputs as efficiently as possible. The SCPT is a visual management tool that was developed to help providers monitor the performance of VMMC service delivery sites and to strategically improve that performance to ensure that sites produce as many quality VMMCs as feasible. Specifically, the SCPT allows VMMC implementers to:Track the key labor and capital inputs to VMMC service delivery;Monitor the numbers of VMMCs performed;Estimate current and optimum site service delivery capacity;Compare VMMCs performed with site targets and current and optimum site capacity;Estimate additional staffing needs to achieve optimum site capacity;Monitor the reach and impact of VMMC demand creation efforts;Compare VMMCs done with number of people reached by VMMC demand creation efforts and number of people seeking services;Track circumcision follow-up visits and number of adverse events;Track uptake of HIV testing, and STI services;Estimate site utilization and site productivity gains/losses.An examination of Manica and Tete provinces in Mozambique, where there was a major strategic restructuring of the program underway and where the SCPT was piloted from September 2016 to September 2017, provides insights into the information that can be obtained from the tool and its potential impact. Although VMMC services were available in Manica and Tete provinces from 2012, coverage remained extremely low by end of 2014. Some progress was made over the next year although coverage remained low. Graphical displays of data using the SCPT were initially analyzed by VMMC implementers in September 2015. However, it took some time to get all stakeholders on board, underscoring the importance of early involvement of end-users in the adoption of the SCPT. Initially, providers thought that the discrepancies between number of VMMCs done and site targets was the result of overly ambitious targets and not differences between number of VMMCs done and site capacity. VMMC implementers in Mozambique began using a VMMC site optimization strategy from September 2016. This strategy was developed based on the significant gaps identified between the number of VMMCs done per site and site capacity given available inputs as well as optimum site capacity given altered inputs. In addition to using the SCPT, the VMMC site optimization strategy involved monthly brainstorming by implementers, development of site-specific targets, and monitoring and regular updating of targets, and continuous service delivery quality improvement.From October 2016, there was a dramatic increase in the number of VMMCs performed in Manica and Tete provinces. The number of fixed service delivery sites providing VMMC services was expanded. In addition, each fixed site extended service delivery by performing VMMCs in outreach sites. This led to significant improvements with current VMMC site capacity increasing and in some cases very close to the site optimum in most sites by the end FY 2017. Importantly, the inclusion of outreach sites also improved access by bringing services closer to harder to reach and lower-income clients. Fixed sites are often located in urban and peri-urban locations and out-of-pocket travel expenses associated with accessing VMMC services can be quite high and lost time and associated income can also be an issue for some .Based on the information site and/or program managers obtained through tracking service delivery using the SCPT, alignment between VMMC site targets and the number of VMMCs performed improved from October 2016, with the number of VMMCs performed much more likely to equal or surpass VMMC site targets at the end of the period. Utilization rates stabilized between October 2016 and September 2017, with VMMCs performed closely tracking VMMC site capacity each quarter in most sites. Both under- and over- utilization were problematic. Factors that influence utilization in addition to site level planning include seasonality, quality and reach of demand creation activities, client access, quality of in-service communication, and client satisfaction with service delivery (e.g. mixing younger and older men may result in older men declining services) , 32. AllIncreases in the numbers of people reached by VMMC services, the numbers of people seeking VMMC services, and the numbers of VMMCs performed over time were observed in half the sites. The fact that the number of people seeking VMMC services was greater than the number of people reached in all sites during most quarters underscores the importance of in-service communications and the potential for strengthening demand creation approaches implemented in Manica and Tete , 39.Envisioned as a planning tool as well, one of the strengths of the SCPT is that VMMC providers can use it to visually represent data on their site’s performance over time. Together with regular reviews of these graphical displays and using the information to recalibrate service delivery, implementers can continuously improve their performance. The power of visual management tools and the role that they can play in improving health facility-level performance is well documented –43. MoreLack of data for site level decision making prompted the development of the SCPT. Before the tool, the site managers had not relevant information on how plan for demand, capacity, quality, and efficiency of the site, focusing only on the use of the achievement vs target as indicator to discuss performance and improvement. With the SCPT, the site managers were able to have information related to target vs capacity vs demand, performance, quality, and efficiency. Being able to precisely plan the number of staff for example base of the site target allow the site manager to bring the site capacity to the level of the target, therefore given them greater opportunities to provide services to all clients who needed it. However, it is not possible to attribute the site improvement and performance to one single tool or interventions. The program benefitted from a baseline assessment  to determine performance against every single standard.As with all monitoring, planning, and modeling tools of this kind, the utility of the SCPT is limited by the amount and quality of data that are entered into it. Even though the data included in the SCPT are routinely collected by sites receiving PEPFAR funding, the quality and level of completeness of the data varies. Another limitation of the SCPT is that it is difficult to compare across sites and identify in what ways well performing sites differ from less well-performing sites. Benchmarking—where performance across sites is compared and top performers are identified and best practices are detected so they can be emulated by peers—is a tool for performance improvement long used in health care and other sectors . An addiBecause the SCPT was built primarily as a site level tool, its principal aim is to pull all relevant data into one repository to support site managers and staff to monitor achievement of VMMC targets and ensure that the resources available at the facility are used to maximize service delivery. However, the tool is also useful for managers above the site level  as they can use the SCPT to obtain insights into program implementation and performance for larger administrative areas. Current discussions about the continued use of the SCPT revolve around identifying the appropriate place for the tool within existing health information systems, such as the District Health Information Software 2 (DHIS2) used in many low and middle-income countries. Given the specificity of the tool both in terms of its programmatic focus and implementation level, Ministries of Health are exploring what technical options may exist to maintain the tool’s use at site level while being able to both pull and push data to the DHIS2. Another option would be to customize the DHIS2 to capture the same inputs and provide same dashboard as the SCPT.While the application of the SCPT in Manica and Tete provinces in Mozambique from September 2016 to September 2017 was not the sole factor underlying the significant scale-up of VMMC service delivery, the use of the tool as part of a broader site optimization strategy showed encouraging results. Workshops and webinars on SCPT have been conducted in Eswatini, Kenya, Lesotho, Malawi, Namibia, South Africa, Tanzania, Uganda, and Zimbabwe in addition to Mozambique. Other countries should consider using the SCPT to improve the performance of VMMC service delivery sites and ensure that they use labor and capital inputs as efficiently as possible to produce as many quality VMMCs as feasible.Finally, the SCPT is a programmatic and implementation tool to improve efficiency and performance of VMMC with the potential strength of using routine data. The operational capabilities of the tool could be adapted and applied to other programmatic areas to drive performance improvements.


Atrial fibrillation (AF) is the most common cardiac arrhythmia in adults. The left atrial appendage (LAA) is the most likely source of thrombus formation in patients with non-valvular atrial fibrillation (NVAF). Left atrial appendage closure (LAAC) represents an effective alternative to NOAC in patients with NVAF. Expert consensus documents recommend intraprocedural imaging by means of either transesophageal echocardiography (TEE) or intracardiac echocardiography (ICE) in addition to standard fluoroscopy to guide LAAC. TEE-guided LAAC usually requires general anesthesia. The ICE technique is a “minimalist approach”, without general anesthesia, but ICE imaging techniques are not yet simplified and standardize, and the ICE may result in inferior image quality compared with that of TEE. Another “minimalist approach” can be the use of ICE via the esophageal route (ICE-TEE), that jet is validated to identify the presence of LAA thrombi in patients and to perform other procedures. In our cath laboratory ICE-TEE to guide LAAC is used in some complex patients. Indeed, our single center experience suggests that ICE-TEE could be a good alternative imaging technique to guide LAAC procedure without general anesthesia. Atrial fibrillation (AF) is the most common cardiac arrhythmia in adults. The prevalence of AF in adults is between 2% and 4%. This percentage significantly increases with age , lifetimIn the treatment of AF patients, together with achieving better symptom control and treating comorbidities, avoiding stroke is of paramount importance, as its consequences are often dramatic .Perhaps, AF has been shown to increase the risk of stroke five-fold; furthermore, AF-related stroke is generally more severe when compared to non-AF-related stroke causing greater disability or worse outcome. Whereas symptomatic burden and AF pattern  are crucial in deciding the anti-arrhythmic strategy, stroke risk has been shown to be affected by the presence of specific risk factors and not by AF patterns, CHA2DS2-VASc is the score most used in clinical practice to identify patients at high stroke risk and to define those who might benefit from oral anticoagulation, evaluating the presence of congestive heart failure, hypertension, age, diabetes mellitus, previous stroke, vascular disease, and female sex ,7,8,9,102DS2-VASc score ≥2 . The implementation of direct oral anticoagulants (DOAC) provided an improvement in the field, as these drugs were proved to be safer and more effective with respect to vitamin-K antagonists (VKA) , with no difference in fluoroscopy time, total procedure time and complications including pericardial tamponade, device embolization and stroke between two groups [ICE has been used for many years to guide structural heart interventions, such as atrial septal defect and patent foramen oval closure, and many interventional electrophysiological procedures involving transeptal puncture. The feasibility and safety of LAA occlusion with ICE guidance have been proved recently. In many centers, it is currently performed as the standard approach for perioperative guidance, despite the need for additional vascular access , as it allows to carry out the procedure under local anesthesia with the patient awake and responsive. If no pre-procedural imaging is feasible, auricular thrombosis might be ruled out using distant contrast injection in LAA under fluoroscopy, with a non-negligible risk of thrombus embolization. For this reason, periprocedural ICE should always be supported by accurate pre-procedural planning using either TEE or CCTA in order to exclude the presence of auricular thrombosis and confirm correct device sizing measurements. Notably, LAA sizing obtained by the combined analysis of ICE images and angiography can slightly vary from measurements obtained with TEE and angiography, even though these differences might be relatively minor with no impact on overall clinical outcomes. Several studies have reported the feasibility and safety of ICE-guided LAAC using these approaches ,45,46,47o groups . In a reo groups . There ao groups . The thiAs a matter of fact, there is a learning curve for ICE-guided LAAC, and this might slow down its use in some centers; in order to overcome such limitations, systematic training of operators, even with the use of dedicated simulators and with dedicated sessions at congresses, might help to let this important technology available in centers performing LAAC. Furthermore, using this technique to guide other procedures, such as septal defect or patent foramen oval closure, might speed the learning curve up allowing the operator to be confident with the methods.Many efforts have been made to develop a new three-dimensional ICE probe in order to achieve better periprocedural guidance with a more accurate evaluation of sizing and of the landing zone, aiming to reduce both periprocedural and long-term adverse events. The use of a novel 3D-ICE probe has been reported, with the ability to provide tilting plane imaging together with 3D-color and spectral Doppler assessment, but currently, such devices are not approved for use in Europe, as they still have not obtained CE certification . ClinicaTEE-guided LAAC usually requires the administration of anesthetic drugs to manage the discomfort of the probe during the procedure and avoid patient movements. Patients referred for LAAC procedures are usually old, and they may have complications related to anesthesia . This is the premise to identify a “minimalist approach”, without general anesthesia, in guiding LAAC. The ICE technique needs minimal sedation, and for this reason, it is the standard in some centers. Instead, the use of ICE via the esophageal route (ICE-TEE) is validated to identify the presence of LAA thrombi in patients with AF, and, when the LAA could be adequately imaged (around 85%), this technique has a sensitivity of 100% and a specificity of 97% .ICE-TEE is concordant with standard TEE for the visualization of the LAA and atrial septal in the vast majority of patients. This alternative use of the ICE probe is safely used to monitor PFO closure without requiring general sedation . MoreoveLAAC currently represents a safe and effective intervention to prevent stroke in AF patients with contraindications to oral anticoagulation or unwillingness to adhere to therapy. Pre-procedural imaging is of primary importance in improving outcomes and should always be carried out either with TEE or with CCTA in order to provide correct sizing measurements and rule out auricular thrombosis. Periprocedural imaging should not be carried out by fluoroscopy alone but should always be accompanied by either TEE or ICE. Even though TEE still remains the gold standard in many laboratories, ICE proved to be safe and effective and can be considered a valuable alternative. TEE-ICE is a new technique, already validated as guidance for other structural heart disease procedures, and clinical trials are needed to validate this interesting imaging technique to guide LAAC. More research is needed in order to move toward a minimalistic approach, aiming to maintain high rates of procedural success while avoiding general anesthesia.


Radiotherapy standardized training (ST) has been conducted for 7 years in China. This investigation evaluated the difficulties of and need for ST of radiation oncology residents (RORs) for gynaecological tumours (GYN) in China.An anonymous online survey was conducted on the “Questionnaire Star” platform. The questionnaire contained 30 questions, including the basic information of the students, their knowledge of radiotherapy theory, training on GYN, the difficulties and needs they faced, and possible solutions.A total of 469 valid questionnaires were collected, resulting in a valid response rate of 85.3%. During the ST, only 58–60% of RORs received training in GYN, with a median clinical rotation time of 2–3 months. Among the RORs surveyed, 50.1% knew the physical characteristics of brachytherapy (BRT), and 49.2% could choose the appropriate BRT for patients. At the end of ST, 75.3% were able to complete the target delineation in GYN independently, and 56% were able to complete the BRT operation independently. The scarcity of GYN patients, insufficient teaching awareness of superior doctors, and lack of interest are the main reasons why ST cannot meet the standard.In China, the ST of RORs in GYN should be strengthened, the teaching awareness of specialist trainers should be increased, and the curriculum should be optimized, especially the curriculum for specialist operation and a strict assessment system. Radiotherapy is the main treatment for common gynaecological malignant tumours such as cervical cancer. A combination of external beam irradiation and brachytherapy is needed in treatment, which involves high requirements for the theoretical knowledge of radiation oncology, radiation biology and physics and the clinical skills of radiation oncologists. However, most medical schools in China do not offer courses in radiation oncology, which makes it difficult for newly graduated residents to master it in the short term.Currently, the training programme of 5 + 3 + X is mostly adopted for radiation oncology residents (RORs) in China; it involves 5 years of undergraduate medical study, 3 years of standardized training (ST), and X years of specialized training . In 2014, radiation oncology (RO) was included in the national ST of residents for a period of 3 years. The main clinical rotation departments currently include the radiotherapy department for 10 months , the general internal medicine department for 10 months , and tumour-related departments for 13 months . At the end of ST, clinical theory and practical ability are assessed with a combination of the daily comprehensive score. Follow-up specialized training has not yet entered the formal implementation stage, but most hospitals have a specialized training mode. According to the requirements of ST, the training time for gynaecological tumours (GYN) is approximately 2 months, but the minimum number of cases is 10. The requirements indicate the need to master the target delineation of GYN and brachytherapy (BRT) operations.In the current ST mode, we conducted a questionnaire survey on radiotherapy ST in GYN to clarify the difficulties and needs related to ST and to explore a better training model.The online anonymous survey was conducted with the help of the “Questionnaire Star” platform, and each participant was limited to completing the survey once. The survey was conducted from January 27, 2022, to February 16, 2022. The respondents were residents specializing in radiotherapy in China. The questionnaire included 30 questions, including the basic information of the students, their knowledge of radiotherapy theory, their training in gynaecological tumours, the difficulties and needs they faced, and possible solutions.The collected data was analyzed using SPSS 19.0 software, and the comparison of variable composition ratios was carried out using the chi-square test.A total of 550 questionnaires were distributed, out of which 528 were received. Fifty-nine respondents who were not residents were excluded, including 15 chief doctors, 41 deputy chief doctors, 1 chief technician, and 2 technicians. Finally, 469 valid questionnaires (85.3%) were analyzed. The collected questionnaires were from 27 provinces, autonomous regions, and municipalities across China. However, there were no data from Qinghai Province, Jiangxi Province, Hainan, Tibet, Taiwan Province, Hong Kong, and Macau. The specific distribution is shown in Fig. Of the respondents, 417 (88.9%) were from tertiary hospitals and 52 (11.1%) were from second-level hospitals. Among them, 392 (83.6%) were from teaching hospitals. Among the hospitals where the RORs worked, 96.4% carried out CT simulation positioning, 25.8% carried out MRI simulation positioning, 95.5% carried out intensity modulated radiotherapy, and 70.6% carried out BRT.Among the 469 RORs, 407 (86.8%) received ST on RO and 62 residents received non-RO ST，including 4 (0.9%) who received ST in obstetrics and gynecology, 43 (9.2%) who received ST in internal medicine, and 15 (3.2%) who received ST in surgery.2 = 0.046), respectively; 79.2% had a rotation period of 1 to 6 months, and half of them had a rotation period of 2 to 3 months.Among all RORs, 327 (69.7%) junior physicians who had been engaged in radiotherapy for 1–3 years received ST on RO and 142 (30.3%) senior physicians who had been engaged in radiotherapy for 4–6 years completed ST on RO. In the two groups, only 192 (58.7%) and 84 (59.2%) underwent GYN rotation in ST  had experience in applicator implantation for patients with GYN. Among them, 48 (27.3%) thought they were skilful at BRT, 81 (46.0%) were familiar with BRT, 34 (19.3%) were generally familiar with BRT, and 13 (7.4%) were not familiar with BRT.Among all participants, 92.1% thought that BRT was very important for patients with locally advanced cervical cancer, 7.7% thought BRT was important, 0.2% thought BRT was generally important, and no participants thought BRT was not important. Only 50.1% of RORs knew the physical characteristics of BRT, and 49.2% thought they could choose the appropriate BRT for patients, as shown in Fig. At the end of the current ST, only 75.3% of RORs can independently complete the target delineation of GYN patients, and 56% of RORs can independently complete the BRT.P = 0.714, χ2 = 0.134).The problems faced by junior and senior RORs in treating patients with GYN are different, as shown in Fig. P = 0.000), as shown in Fig. In practical clinical work, when residents encounter problems, junior physicians usually prefer to consult their superiors, textbooks or professional books, periodicals, or literature to solve the problems. The proportion of junior physicians who consulted their superiors was significantly higher than that of senior doctors  in the incidence of female malignant tumours, uterine cancer ranks eighth , and cervical cancer ranks seventh in the mortality rate . The incidence and death from cervical cancer are still on the rise [RORs still prefer the help of superior doctors to solve clinical problems, so the teaching awareness of superior doctors needs to be improved. In daily teaching, multiple forms of teaching modes should be combined , 5, 7, 8The setting of specialized courses is a common requirement in the ST of RORs, such as imaging, radiation physics, and biology . BrachytThis survey was aimed at China’s radiation oncology residents, with widely distributed data sources and a valid questionnaire response rate of 85.3%, making the conclusions generally applicable nationwide. However, there were some limitations to this survey. Since the main objective was to investigate the standardized training mode in gynecological tumor subspecialties for radiation oncology residents in China, it is unfortunate that no detailed investigation was conducted on brachytherapy. In the future, we plan to investigate on the relevant details of brachytherapy.The ST of the gynaecological oncology subspecialty of radiation oncology in China has not been fully popularized, and residents’ mastery of specialized skills and theories still cannot meet the training requirements. It is necessary to increase the teaching awareness of specialized training teachers and continue to optimize the curriculum, especially by improving the curriculum for specialized operation and strict assessment systems.