In addition, the metabolic coordination between leaves and grains, which is crucial for crop responsiveness to elevated CO2, has never been examined closely. Here, we address these two aspects by multi-level analyses of data from several free-air CO2 enrichment experiments conducted in five different countries. There was little effect of elevated CO2 on yield (except in the USA), likely due to photosynthetic capacity acclimation, as reflected by protein profiles. In addition, there was a significant decrease in leaf amino acids (threonine) and macroelements (e.g. K) at elevated CO2, while other elements, such as Mg or S, increased. Despite the non-significant effect of CO2 enrichment on yield, grains appeared to be significantly depleted in N (as expected), but also in threonine, the S-containing amino acid methionine, and Mg. Overall, our results suggest a strong detrimental effect of CO2 enrichment on nutrient availability and remobilization from leaves to grains.While the general effect of CO 2 on grain yield, and reveals relevant modifications in N and Mg metabolism.A study of wheat grown at five FACE facilities around the world shows a low effect of CO Bread wheat (Triticum aestivum L.) constitutes up to 95% of global wheat production, while durum wheat (Triticum turgidum ssp. durum) makes up the remainder  by 2100  conditions and inter- and intra-specific variability. While a CO2-driven increase in yield is generally observed, there is substantial variation, with sometimes no yield gain at all  leads, on average, to a yield enhancement of only 10% , lower photosynthetic capacity (maximum carboxylation rate Vcmax), and/or lower ribulose 1,5-bisphosphate regeneration ability  compared with rain-fed plants (up to 10%), although yield absolute values are, as expected, lower under water restriction  conditions  have shown that the Rubisco content did not increase, while there was a slightly higher content of Rubisco activase (2 loss)  content, less protein, and more starch and fibres  and free hexoses (fructose and glucose) but less sucrose and alanine, regardless of developmental stage. At maturity, grains are generally depleted in free amino acids, except for the branched-chain amino acids valine, leucine, and isoleucine, which are significantly more abundant  produced at elevated CO2 likely depends not only on the cultivation conditions , but also on soil properties and the species and cultivar of wheat. For example, wheat lines of different ploidy do not respond similarly to elevated CO2 in FACE experiments, with hexaploid bread wheat being the most responsive in terms of yield (but the least in terms of photosynthesis)   conducted in different countries  with different species (T. aestivum and T. turgidum ssp. durum) and cultivars  and grain biochemical composition. Our working hypothesis was that (i) detrimental effects of elevated CO2 on photosynthesis and N assimilation were associated with significant changes in protein and amino acid composition in leaves, and thus (ii) the amino acid composition of grains was affected, reflecting alteration of both the provision and recycling of nitrogenous compounds; and (iii) there might be a relationship between grain metabolic features and yield.Taken as a whole, whenever there are differences among growth conditions and cultivars, it is difficult to understand the physiological mechanisms underlying the response to elevated COultivars . We carr2 conditions. FACE facilities included three to five rings (for each CO2 concentration condition) with a diameter of 12–14 m each or (in Stuttgart-Hohenheim) 2×2 m square plots. In all cases, CO2 enrichment was performed from sunrise to sunset  or 24 h a day  and throughout the entire growing season. The principle of operation and the performance of the FACE systems used here have been described previously , grain samples were collected for agronomic and metabolic analyses.The experiment was conducted with durum wheat  and bread wheat . The study involved five FACE facilities located in Stuttgart-Hohenheim (Germany), Fiorenzuola , Changping-Beijing (China), Horsham , and Beltsville (USA), in which plants were exposed to ambient (400 ppm) and elevated (550 ppm) CO2 conditions. The light-saturated rate of CO2 assimilation (Asat), stomatal conductance (gs), and intercellular CO2 concentration (ci) were estimated at a photosynthetic photon flux density of 1500–1600 µmol m−2 s−1 using equations developed by 2 response curves were constructed by a series of measurements whereby photosynthesis was determined first under standard conditions (400 µmol mol−1), then at low CO2, then back to standard conditions, and then at high CO2: overall, the sequence of CO2 concentrations in the reference channel was 400, 200, 100, 50, 200, 400, 600, 750, and 950 µmol mol−1. Estimations of the maximum carboxylation velocity of Rubisco (cVmax) and the maximum electron transport rate contributing to ribulose 1,5-bisphosphate regeneration (Jmax) (measurements made in Germany) were made using the method of Analyses were carried out in healthy expanded flag leaves grown under ambient or elevated COC and N concentration (%) analyses were determined using an elemental analyzer . Micronutrient and macronutrient concentrations were determined by inductively coupled plasma/optical emission spectrometry .13C=(Rsample/Rstandard)–1 and expressed in ‰, where Rstandard is the 13C-to-12C ratio of the international standard (V-PDB). The carbon isotope discrimination (Δ) was calculated as follows:Carbon isotope composition was determined using an elemental analyzer  coupled to an isotope ratio mass spectrometer . Values were expressed as delta values δ 13Cair (‰) is the isotope ratio in atmospheric CO2. The addition of  CO2 in the course of the FACE experiments was such that δ 13Cair was lower than the natural 13C abundance in the atmosphere (near –8‰). The δ 13C value of the added CO2 was known (for the experiment in Germany) or calculated from mass balance, taking advantage of the known contribution of added CO2 to total CO2: c=(550–400)/550=27.2%. In fact, if the isotope fractionation under ambient CO2 and under FACE conditions was the same, then the proportion of C coming from the added CO2 could be calculated as c=/, where OM refers to organic matter and amb refers to ambient conditions, and δ 13C values are abbreviated to δ to simplify the notation. δ* OM,FACE is the δ 13C value of organic matter under FACE conditions if the fractionation had not changed compared with ambient conditions. In practice, the isotope fractionation could have varied, and thus (neglecting denominators): δ* OM,FACE =δ OM,FACE+Δ FACE–Δ amb, where Δ FACE and Δ amb are the net photosynthetic fractionation under FACE and ambient conditions, respectively. These equations can be combined to give c=/–(Δ FACE–Δ amb)/. The right term is relatively small since added industrial CO2 was naturally 13C depleted and thus δ CO2,amb–δ CO2,added is much larger than Δ FACE–Δ amb. That is, by neglecting the right term, δ CO2,added can be estimated (and therefore Δ FACE with equation 1) by imposing c=0.272. Note that such an approximation was not critical, since even under the assumption (Δ FACE–Δ amb)/ represented 0.10 (i.e. 40% error in c); this would ultimately cause an error of only 1‰ in Δ FACE.where δ Extraction, purification, and quantification of abscisic acid (ABA) were carried out as described by Sucrose, glucose, and fructose content were determined using a Beckman P/ACE5500 capillary electrophoresis system , following the method of Amino acids were derivatized at 22–25 °C for 12–16 h with 1 mM fluorescein isothiocyanate dissolved in 20 mM acetone/borate, pH 10. The content of single amino acids was determined by capillary electrophoresis in a Beckman-Coulter PA-800 system.Samples were previously quantified by microBCA analysis (Pierce) and similar amounts (5 µg per sample) were individually dissolved in 8 M urea, 25 mM ammonium bicarbonate, reduced with DTT, and alkylated with iodoacetamide, according to a method described by 2, univariate statistics were conducted with a two-way ANOVA, with one factor representing ‘conditions’  while the second factor was CO2. It was not possible to carry out a three-way analysis because not all species/cultivars were represented in all countries. Thus, here, the two-way ANOVA was constructed with the factor ‘conditions’ having 12 possible qualitative values (combinations time–country–cultivar) and the factor ‘CO2’ having two values (ambient or elevated). Unless otherwise stated, statistical significance was accepted when P<0.05. Multivariate statistics were carried out using orthogonal projection on latent structure , using CO2 as the predicted Y variable , and metabolic features  as predicting X variables. Before running the OPLS, a principal component analysis was conducted to check the presence of outliers (samples outside the Hotelling’s ellipse). The performance of the OPLS was assessed using the correlation coefficient between predicted and observed Y (R2), the cross-validated correlation coefficient (Q2), the Q2 intercept of the permutation test (which was checked to be negative), and the P-value of testing the OPLS model against a random-error model (i.e. average ±error) via a χ 2 test . Univariate and multivariate analyses were combined using –log (univariate) plotted against the OPLS loading (pcorr) in a volcano plot. To explore the relationship between yield and grain composition, we conducted (i) an OPLS using yield as the predicted Y variable (with the same parameters of performance as described above) and (ii) a univariate analysis by linear regression (done in R). Linear regression was done without and with variable elimination. Without variable elimination, each variable was correlated with yield separately and the associated P-value is reported. To carry out variable elimination, we used a multiple linear regression. It was carried out via sampling subsets (regsubsets) of eight variables (304 iterations) and a correlation plot was generated to select the eight most correlated variables overall. Then, a multiple linear regression model (lm) comprising only these eight variables was generated and the P-value for each variable was calculated.To explore the effect of CO2 mole fraction (ca) used during growth  in Australia, plants generally had lower photosynthetic rates compared with other countries. These effects were unrelated to stomatal conductance  (ci/ca <0.7) and in Australia (ci/ca ≈0.7) at elevated CO2. The natural carbon isotope abundance (δ 13C) in raw leaf matter was measured, and ‘average’ ci/ca (i.e. across the leaf lifespan) was estimated using the simplified equation of 12C/13C fractionation (raw matter versus source CO2) was within 20–22‰, showing that average ci/ca was always ~0.7. However, the isotope fractionation was significantly lower (and therefore so was average ci/ca) in Germany, Italy, and Australia at elevated CO2, suggesting a long-term decrease in stomatal  conductance and/or an increase in photosynthetic capacity at elevated CO2. In addition, there were no significant changes either between CO2 conditions or among countries in ABA content (data not shown).The photosynthetic activity of flag leaves was assessed using measurements of gas exchange at various times of the development cycle (shown as BBCH stage numbers), at the COg growth . Overalloperties : (i) in ductance , which w (ci/ca) , which w2 are represented as a volcano plot in 2 and ‘conditions’ . Growth at elevated CO2 led to an increased content of several elements  and a decrease in K. Several enzymes  had a lower content at elevated CO2, and so had threonine. There was a tendency to have more fructose and less sucrose at elevated CO2 (significant changes in ANOVA) but OPLS loadings were rather small, showing that the change in sugar composition was not a strong marker of growth at elevated CO2. When expressed as a percentage of total amino acids and proteins, glycine and glycine decarboxylase (GDH subunit h) were lower (P<0.001) under elevated CO2 . This phenomenon was clearly visible in the results of the two-way ANOVA, where several features appeared to be significant for the CO2 × conditions effect . A statistical analysis combining multivariate (OPLS) and univariate (two-way ANOVA) statistics was performed and results showing the effect of COated CO2 , likely ated CO2 , and thrated CO2 , showings effect . For exad with K . Howeverated CO2  followed2. By contrast, there were significant decreases in several elements , amino acids , and the glutamate derivative γ-aminobutyrate (GABA). Although nitrogen content (%N) was found to be lower, there was no significant change in protein content. Such a decrease in N content could be explained by changes in the concentration of many N-containing compounds , which in total sum up to a larger and significant change in overall %N. When plotted together, %N and carbon content (%C) showed considerable scattering across countries  . As in leaves, an analysis combining univariate (ANOVA) and multivariate (OPLS) statistics was performed and presented as a volcano plot . Interesountries , with a nd Italy , D. Mores effect . Elevate and Mg) . In factated CO2  was not −2) and high values in Australia and Italy (≈600 g grain m−2) (−2). To gain insight into the metabolic determinants of yield, we conducted both a multivariate OPLS analysis using yield as a predicted quantitative Y variable, and a univariate analysis by linear regression. The OPLS analysis generated a good statistical model (R2=0.75) with good robustness (Q2=0.67) and very high significance (PCV-ANOVA=10–16). When observed and predicted yield were plotted together, it was clear that the relationship was driven partly by differences between countries  (P<0.05) but their loading value was close to zero, showing that their impact  was numerically very small. When plotted separately, there was a positive relationship between yield and the glutamine-to-valine ratio in grains  . The difain m−2) , and soiain m−2) , or sowiountries  and not reatment . Accordi the USA . The bes the USA  is assoc the USA  incorporscarded) . Regardlscarded) , C. Whenscarded) . Tyrosinn grains .2 in leaves and grains. Our results show that elevated CO2 (i) had a limited effect on photosynthesis rate and grain yield, reflecting photosynthetic acclimation; (ii) caused a decline in proteins involved in photosynthesis, photorespiration, or N assimilation in leaves; and (iii) altered grain quality, with lower contents of amino acids and mineral nutrients. In addition, a quantitative analysis of yield suggested that high yield values correlate with higher glutamine, K, and Mg contents and lower valine content.Here, we used five FACE sites in different countries, with different wheat cultivars/species, to investigate the metabolic effects of elevated CO2 depend considerably on surrounding  environmental conditions. Here, we found that with the exception of the FACE site in the USA, grain yield was not significantly affected by elevated CO2. In practice, changes in wheat yield in response to high CO2 are determined by the cultivar(s), fertilization protocols, and interactions with other environmental factors such as water availability and temperature. The absence of an effect of CO2 was probably linked to the lack of significant changes in assimilation (mechanisms are further discussed below). Accordingly, several previous studies have shown that the initial stimulation by CO2 can be compensated for by acclimation  and the carboxylation capacity, which is linked to leaf N content. Here, the absence of significant differences in stomatal conductance (accompanied by the lack of an effect on leaf ABA content) suggests that stomatal limitation of CO2 diffusion was a minor component of acclimation under our conditions. The lower content of nitrate reductase (and other proteins) suggests an inhibitory effect of elevated CO2 on N metabolism, as previously observed in wheat leaves , our study shows important differences in yield between locations and cultivars. The lowest values were found in the USA, where sowing density (120 m−2), precipitation, and cumulated day·°C values were low  despite low plant sowing density and low cumulated days∙°C; this suggests that perhaps the two most important factors for CO2 responsiveness were available N and intercepted light  under our conditions. Differences between locations could also have originated from the contrasting behaviour of the two species of wheat studied, with bread wheat exhibiting generally higher yield values than durum wheat. This species effect comes from the fact that durum wheat is more water conservative and thus more suitable to grow in stressful environments, while bread wheat is believed to have a higher yield potential . Here, photosynthesis increased by a modest but significant amount at elevated CO2  or did not increase at all . In Italy, the lack of a stimulating effect of CO2 mole fraction was associated with a decrease in stomatal conductance  (A/ci response curves for wheat grown in Germany suggest a lower Vcmax (non-significant) and Jmax (P<0.05) ; as a result, the fructose-to-sucrose ratio was larger at elevated CO2 in two countries   . In addiraction) , and A/c(P<0.05) . More ged Italy) . In othe2 had a clear effect on grain composition, leading to lower contents of N, amino acids, and microelements (2 on N content (and proteins) in grains has been documented before, and has been suggested to be linked to perturbations in both leaf-to-grain N transport (e.g. glutamine) and amino acid metabolism in grains . Nutrient quantitation during grain filling in different cultivars of spring wheat has suggested that Mg is remobilized from the lower stem and leaves to grains, with little effect of cultivar  carboxylase fixation , alanine comes from pyruvate, and the biosynthesis of aromatics requires PEP. Therefore, it is possible that the decrease in amino acids was caused by a specific effect of elevated CO2 on (phosphoenol)pyruvate synthesis via glycolysis. For example, pyruvate Pi dikinase, which resynthesizes PEP from pyruvate, has been shown to be essential for starch accumulation at the late grain-filling stage  in Australia  to delineate the effects of elevated CO2; in particular, soil composition (and not only N fertilization) seems to be important. Yet, the decrease in threonine and Mg content in grains was independent of ‘conditions’, that is, it was not associated with a CO2 × conditions interaction effect.Importantly, these metabolic effects depended on ‘conditions’, that is, country and species/cultivar . It was location . Similarustralia , where Nustralia , while tustralia . There w2 was not a confounding factor, since there was no significant effect of CO2 on yield overall  and to alter grain composition, in particular %N, amino acids , and minerals (Mg). The yield was related to metabolic features of grains but this relationship was not influenced by elevated CO2.Taken as a whole, our results show that growth at elevated CO2 fertilization can be explained not only by the down-regulation of carbon fixation  but also by an effect on grain metabolism itself. In particular, elevated CO2 appeared to be detrimental to N metabolism, with a decrease in several amino acids and, accordingly, a positive effect of elevated CO2 on yield was observed only in the USA, where N fertilization was the highest.The lack of a positive effect of CO2 also had an effect on Mg redistribution between source leaves and grains. The detrimental effect of CO2 on the content of microelements has been found elsewhere for Zn  to affect yield. In addition, the relationship between Mg content in grains and yield is not driven by genotype (25Mg (or 26Mg) would be helpful to examine the role played by phloem Mg transport during remobilization from leaves at elevated CO2.Elevated COor Zn  would be necessary to determine the best solutions to improve yield responsiveness to CO2. In addition, the selection of varieties with a high harvest index, which is associated with optimal allocation and efficient nutrient remobilization capacity, might be desirable to obtain better grain yield and quality at elevated CO2.The overall effect on N, K, and Mg nutrition found here raises the question of whether specific fertilization management strategies could compensate for the lack of effect of elevated CO13C-valine and isotope-assisted metabolomics. This will be addressed in a future study.Finally, we found that wheat grain yield was related to glutamine and valine grain content, regardless of country and cultivar, showing the importance of amino acid metabolism for grain maturation. However, while the role of glutamine is clear , understanding the metabolic role(s) of valine requires further work, such as tracing with JXB online.The following supplementary data are available at Fig. S1. Elemental composition of soil (top layer) at the different sites used in this study.2 × conditions  effect. Fig. S2. Leaf features significant for a CO2 × conditions  effect. Fig. S3. Grain features significant for a COFig. S4. Correlation plot showing the best variables obtained by subset sampling in linear models of yield.Fig. S5. Photosynthetic response curve for wheat cultivated in Germany.eraa330_suppl_Supplementary_Figures-S1-S5Click here for additional data file.


The authors wish to make the following corrections to this paper :On page 14, in paragraph two of Chapter 4, Echoviruses, the sentence: “The United States, much of the European Union and Japan found a lack of experimental evidence to approve Rigvir in cancer treatment [99]” should be deleted. On page 15, line one, the sentence: “One such retrospective study showed a statistically significant increase in 3-year survival of melanoma patients when treated with Rigvir post-surgery than surgery alone or with other immunomodulators [97]”, should be changed to: “One such retrospective study showed a statistically significant 4.39–6.57-fold decrease in mortality of melanoma patients when treated with Rigvir post-surgery than surgery alone [97]”. On page 15, line three, the sentence: “For best efficacy, Rigvir is injected intratumourally rather than intramuscularly, increasing 5-year survival between 29.9% and 19.5% [97]” should be deleted.On page 15, paragraph two, line seven, the sentence “In the case of the stage IV histiocytic sarcoma patient, Rigvir was administered as part of a therapeutic cocktail including multisite radiation therapy, doxorubicin, cyclophosphamide and helixor P” should read: “In the case of the stage IV histiocytic sarcoma patient, Rigvir was administered for about 2 years followed by multisite radiation therapy, doxorubicin, cyclophosphamide and helixor P.”On page 15, paragraph three, line two, the sentence “One patient with a basal cell carcinoma, a diagnosis with a median expected survival of five months, was treated with Rigvir post-surgery [101]” should read: “One patient with melanoma unknown primary brain metastasis, a diagnosis with a median expected survival of five months, was treated with Rigvir post-surgery [101].”The authors would like to apologize for any inconvenience caused to the readers by these changes.


True lumen flow was streamlined, and the overall fluid velocity and pressures decreased . This protocol provided a standardized method to evaluate the effects of MFM treatments in TBAD on geometric analyses, PMC, and CFD outcomes.Recently, multilayer stents for type B aortic dissections (TBAD) have been proposed to decrease false lumen flow, increase and streamline true lumen flow, and retain branch vessel patency. We aimed to provide a protocol with standardized techniques to investigate aortic remodeling of TBAD by multilayer flow modulators (MFM) in static geometric and hemodynamic analyses. Combining existing literature and new insights, a standardized protocol was designed. Using pre- and postoperative CT scans, geometric models were constructed, lumen dimensions were calculated, computational fluid dynamics (CFD) models were composed, and velocity and pressures were calculated. Sixteen TBAD cases treated with MFM were included for analysis. For each case, aortic remodeling was analyzed using post-processing medical imaging software. After 3D models were created, geometrical anatomical measurements were performed, and meshes for finite element analysis were generated. MFM cases were compared pre- and postoperatively; true lumen volumes increased ( Medical treatment is still the preferred treatment option in many centers for the treatment of acute type aortic B dissection (TBAD). However, pharmacological treatment fails to induce aortic remodeling with progression to aneurysm formation requiring aortic intervention within seven years in nearly 40% . Now accHowever, stent graft-induced new entry tears into the distal thoracic or abdominal aorta can arise as a complication of the TEVAR during the treatment of acute TBAD ,4. Besid® Cardiatis, Isnes, Belgium), which is a self-expanding cobalt alloy bare-metal stent with interrelated wires in three to five layers. MFMs have been designed to be used in complex endovascular aortic repairs, mainly for thoracoabdominal aneurysms. Specific characteristics of the MFM stents include (i) laminar blood flow with less wall shear stress; (ii) no necessity for extra procedures to vascularize branch arteries; (iii) possibility to overlap to form complex structures; (iv) high resistance to kinking and fatigue; (v) the possibility of custom MFM construction to match patient’s anatomy [Another alternative to traditional TEVAR is the multilayer flow modulator stent . The research was carried out in accordance with the Code of Ethics of the World Medical Association (Helsinki Declaration) of 1975, as revised in 1983. The implementation of the presented study was supervised by the Ethical Committee of the Lucian Blaga University of Sibiu and by the Ethical Committee of Polisano European Hospital . Starting from May 2018, the new rules defined in the EU General Data Protection Regulation (GDPR) were embraced and respected in the study.From a prospective single-center MFM registry cohort, the anonymized CT scans of 16 patients with TBAD who underwent endovascular repair using MFM stent between April 2014 and February 2018 were consecutively included in the Polisano European Hospital, Sibiu, Romania. CT scans were collected preoperatively, directly postoperatively, and at the latest follow-up moment (varying from 1 to 36 months postoperatively). A study protocol was formulated by the authors to perform geometric analyses of TL and FL, PMC, FLI, and CFD.Inclusion criteria in the study were: (i) primary type B aortic dissections and residual type B dissections following type A dissection repair; (ii) type B aortic dissections identified by angio-CT scan ; (iii) patient age between 18 and 85 years; (iv) non-pregnant patients; (v) patient was available for follow-up.Although the use of the MFM for aortic dissections is outside instruction-for-use (IFU), the IFU, as established for aortic aneurysms, was utilized. These included: (i) a minimum two-centimeter landing zone in non-diseased aortic wall proximally and distally to the intended placement zone of the MFM; (ii) aneurysm diameter below 65 millimeters, if present; (iii) MFM oversizing of 15–25% in the landing zones compared to the aortic wall; (iv) in overlapping stents, the proximal stent should have a smaller diameter and should be deployed first. Hereafter, the larger distal stent was deployed. The overlap of the stents should be at least six centimeters for straight aortas and eight centimeters for angulated aortas.CTA scanning was performed in the supine position using a 128-row multi-slice scanner . CT scans covered the full extension of the dissection. DICOM files were obtained with a slice thickness of 0.6 mm and a pixel size of 0.625 × 0.625 mm. Surface reconstruction of the aortic dissection was accomplished using MIMICS  postprocessing software. Within MIMICS, a standardized protocol for image segmentation was performed for each CT scan to create 3D aortic models representing the false and true lumen. The contrast-enhanced CT scans in digital imaging and communications in medicine (DICOM) format were imported into MIMICS. Firstly, the Hounsfield Units threshold was set at a minimum of 175. This threshold functionality offers clear visualization of contrast-enhanced anatomy (thoracic aorta and bone) by allowing the low-attenuation anatomy (such as soft tissue) to be removed.Consequently, automated software functions, including “Region growing”, “Calculate 3D”, “Crop Mask”, “Edit Mask”, and “Dynamic Region Growing”, were executed. Hereafter, the aorta was isolated A–C, and True and false lumens were distinguished in MIMICS based on colors  to demonstrate the dimensions of both lumens. Hereafter, the geometrical evaluations of true and false lumen volumes, true lumen diameters at PMC, and FLI were performed pre- and post-procedurally and during the latest follow-up. FLI was calculated using the following formula, by dividing the false lumen volume to the volume of the true lumen.The geometrical reconstruction of the aorta was transferred to 3-Matic  post-processing software to create a clean 3D model. Entry and re-entry tears between true and false lumens were created in 3-Matic conform contrast continuation, and size as observed in the CTA. Subsequently, an STL-file was created.CFD modeling was used to study hemodynamics in aortic dissections since flow, turbulence, and blood-wall-endoprosthesis interactions play a key role in the pathology and treatment of aortic dissections. Therefore, the STL-file was imported in Ansys Workbench . After using the “Generate Part Names” function, aortic geometry was shown as “Solid Full Display”. Next, the surface was altered using the “Segment/Trim Surface” option and “By Angle Method” with an angle of 35°. The most proximal part of the aorta was identified as the “Inlet” in the “Create Part DEZ”, while all the branches were identified as “Outlets” in the “Create Part DEZ”. Afterward, curves were created using the “Extract Curves from Surfaces” option. The aortic wall itself was deselected, while the inlet and outlet surfaces were selected using the “Select All Appropriate Visible Objects” option. The “BSpline” function was chosen, and new curves were created. Now, for the aortic wall, the option “WireFrame Simple Display” was used. Next, using the “Create Body” option, fluid  was added to the model. The “Centroid of 2 Points” function was selected, and, using the mouse, two 3D locations within the aortic wall were selected to instruct ANSYS to create the fluid part. Subsequently, the aortic wall was again fully displayed, using the “Solid Full Display” option. Now the “Global Mesh Size” option was used, including the following configuration: “Scale Factor” = 0.5, “Max Element” = 2, “Curvature/Proximity Based Refinement” should be enabled with “Min. Size Limit” = 0.5, “Elements in Gap” = 1, and “Refinement” = 18. The remainder of the parameters were retained in default settings.Now, the mesh was created using the “Volume Mesh” option. The next settings were used: “Mesh Type” = “Tetra/Mixed”, “Mesh Method” = “Robust (Octree)”, and “Create Prism Layers” was disabled. Following, the “Compute” button was chosen. The mesh was now examined using “Surfaces” and “Solid and Wire” functions. The prism layer mesh contained orthogonal prismatic cells, normally reside next to wall boundaries. The geometry and mesh were exported in “Select Solver” under “Output” using the following settings: “Output Solver” = “ANSYS Fluent”. In “Boundary Conditions” under “Output”, the right boundaries were selected: aortic wall = “Wall”, proximal aortic inlet = “Velocity-inlet”, fluid or blood = “Fluid”, and distal branches = “Pressure-outlet”. Finally, the aortic flow model was exported, and the link between ICEM CFD and Fluent Set-up was created.3 and overwriting the specifications of the fluid. The “Inlet Velocity” was set at 0.35 m/s , and the “Outlet Pressure” at 0 Pa (a steady-state was used). Altogether, the following “Reference Values” were entered: “Area”: 1 m2, “Density”: 1060 kg/m3, “Enthalpy”: 0 j/kg, “Length”: 300 mm, “Pressure”: 0 pascal, “Temperature”: 309.15 K (=36 °C), “Velocity”: 0.35 m/s, “Viscosity”: 0.0035 kg/m-s, and “Ratio of Specific Heats”: 1.4. The calculations were running with 10 static iterations, and, for the Solution, another 10 static iterations were run. The results of the Solution were linked from the Fluent menu. To represent overall fluid velocity and overall fluid pressure, Streamline and Contour functions in ANSYS were used, respectively.In “Set-up”, “Scale” was set at 0.001 (to convert meters into millimeters) for “X, Y, and Z Scaling Factor”, and default settings were retained for the remainder of parameters. The specifications of the fluid were converted from default air into the blood, by changing “Density” to 1060 kg/mAll segmentation steps, geometrical reconstruction, and CFD analyses were performed using a high-speed workstation ; RAM Memory: 32GB; and Video Card: NVIDA Quadro M4000 (8GB GDDR5 + 16GB Shared Memory).p-values <0.05 were considered statistically significant. For the presentation of the cases: continuous variables were expressed as mean ± standard deviation when normally distributed and as median and interquartile range when skewed. Categorical variables were expressed as percentages. Wilcoxon signed-rank test was used to compare preoperative and postoperative outcomes. Of all variables, a percental difference between pre- and directly postoperative was calculated. Hereafter, the two-tailed Spearman Rank Test was performed to assess the relationship between geometric and CFD results. All analyses were performed with SPSS . The In this study, CT scans of 16 cases were included in for which the MFM stent was used as an intervention for TBAD. These 16 patients with complicated TBAD underwent endovascular repair using new generation MFM stents: two patients acute TBAD, six patients sub-acute TBAD, and eight patients chronic TBAD. All MFMs were placed in the true lumen. Detailed 3D reconstructions of the primary three cases with the longest follow-up are presented in 3 (IQR 34.95–110.89 cm3) preoperatively to 83.26 cm3 (IQR 111.31–194.57 cm3) directly postoperatively (p < 0.001). Overall, a mean increase of 157% was observed. During the latest follow-up visit, the median true lumen volume increased to 174.17 cm3 . Compared to the preoperative situation, a mean increase of 201% was observed. In all studied cases, true lumen volume increased at the latest follow-up, with a maximum increase at case no. 9 (954%) and a minimum increase at case no. 13 (140%). 3 (IQR 143.76–299.49 cm3) preoperatively to 133.69 cm3 (IQR 83.43–224.91 cm3) directly postoperatively (p < 0.001) was observed, with an overall mean decrease of 26%. During the latest follow-up visit per case (median follow-up 12 months), the median false lumen volume decreased to 123.42 cm3 . When comparing the total volume of the aorta for the preoperative and postoperative, a mean decrease of 7.21% was observed; while comparing the preoperative and latest follow-up, a mean decrease of 17.36% was observed see .p < 0.001). Overall, a mean true lumen increase of 374% was observed. During the latest follow-up visit, the median true lumen diameter at the PMC increased to 2.18 cm . Compared to the preoperative situation, a mean increase of 412% was observed.True lumen diameter at the PMC was compared pre- and postoperatively per case D. A medip < 0.001) directly postoperatively. Within the 16 studied cases, FLI decreased for all cases, with a mean value of 60%. p = 0.008). Compared to the preoperative situation, a mean decrease of 67% was observed. Case no. 1 presented the maximum decrease of FLI ratio during the latest follow-up (100%), and case no. 7 the minimum decrease (27%).Examination of the FLI ratio demonstrated a median decrease of 2.68 (IQR 1.46–6.56) preoperatively to a median value of 0.92  postoperatively. A median percentage decrease of overall fluid pressures was observed of 66.7%  postoperatively (ratively B.p = 0.014) and a strong negative correlation between true lumen volume change and FLI change . Furthermore, moderate negative correlations were found between false lumen volume change and true lumen diameter at PMC change  and false lumen volume change and CFD pressure change . A negative correlation was also found between true lumen diameter at PMC change and FLI change , and a moderate positive correlation was found between true lumen diameter at PMC change and CFD pressure change .The correlation analysis of preoperative and directly postoperative geometric and CFD results showed a strong positive correlation between true lumen volume change and true lumen diameter at PMC change  is clinically relevant in the light of hemodynamic alterations and wall stress . ResultsThe concomitant increase of intraluminal flow and decrease of turbulent flow  in false lumen was suggested to induce positive shears stress, which could lead to endothelialization in the MFM and co-occurring of thrombosis of the false lumen. Interestingly, this theory is supported by the fact that these processes progress in the postoperative period ,27,28. TAlthough the group was small, the positive and negative outcomes in the correlation analyses illustrated: (i) the beneficial effect of measuring the alterations in the diameter at the PMC as it correlated with both false and true lumens; (ii) the simultaneous enlargement of the true lumen and decrease of the false lumen as an effect of the endovascular procedure; (iii) the relationship that could be found between the geometric and CFD analyses.The assessment of true and false lumen diameters and measured volumes showed an increase, respectively decreased, in the percent compared for each patient to the preoperative outcome, postoperative outcome, and the latest follow-up. The length of the longest follow-up varied for each patient from 1 month to 36 months see .Analysis data from The main aim was to produce a standardized protocol to analyze aortic remodeling in TBAD. However, we were aware of the limitations by presenting our geometrical analysis and hemodynamic changes after MFM stents placement in TBAD in this small case series. A discussion can be made that it could be only the mechanical effect of the expansion of the true lumen by a “bare” stent of any type, as we did not have a control group or other analyses of treatment with covered stents or no stents. Therefore, we encourage others to use this standardized protocol to compare data, as the protocol would be applicable for evaluations of other endovascular devices as well. The segmentation software was used by bio-engineers to analyze the geometrical cardiovascular anatomy and compute parameters. A limitation, specifically considering the protocol, is the lack of scientific input from different experts from different fields and backgrounds, as could be organized as a Delphi method discussion. Nonetheless, the current protocol could already be used to evaluate the efficacy of endovascular devices and might be improved in future versions. Potential changes in future work might include: (i) changes in the geometry of the aorta could be investigated using the prism layers for the mesh exports inside the ANSYS software; (ii) a transient state will be studied instead of steady-state for outlet pressure. The relatively short and variable follow-up length demands further studies with larger patient groups, followed over a longer period. Additionally, in future studies, alternative analysis methods, such as finite element analysis and computational fluid dynamics, should be undertaken. The authors conclude that the current protocol can be used to evaluate the effects of the multilayer stent and potentially other endovascular devices on geometric analyses, PMC, and both qualitative and quantitative CFD outcomes. Further research is needed to evaluate the efficacy and clinical effects of multilayer stents used for the endovascular treatment of thoracoabdominal aortic dissections.


Estrogen receptors (ERα and ERβ) are ligand-activated transcription factors that play different roles in gene regulation and show both overlapping and specific tissue distribution patterns. ERβ, contrary to the oncogenic ERα, has been shown to act as an oncosuppressor in several instances. However, while the tumor-promoting actions of ERα are well-known, the exact role of ERβ in carcinogenesis and tumor progression is not yet fully understood. Indeed, to date, highly variable and even opposite effects have been ascribed to ERβ in cancer, including for example both proliferative and growth-inhibitory actions. Recently ERβ has been proposed as a potential target for cancer therapy, since it is expressed in a variety of breast cancers (BCs), including triple-negative ones (TNBCs). Because of the dependence of TNBCs on active cellular signaling, numerous studies have attempted to unravel the mechanism(s) behind ERβ-regulated gene expression programs but the scenario has not been fully revealed. We comprehensively reviewed the current state of knowledge concerning ERβ role in TNBC biology, focusing on the different signaling pathways and cellular processes regulated by this transcription factor, as they could be useful in identifying new diagnostic and therapeutic approaches for TNBC. Estrogen receptors (ERs) are ligand-activated transcription factors that mediate the effect of estrogens in the development and growth of both normal and malignant mammary tissues. Ligand-activated ERs are able to form dimers that, directly or through other proteins, bind specific estrogen response elements (EREs) in the target gene promoters and regulate their transcription ,2.ER alpha (ERα) is the major driver of ~75% of breast cancers (BC), its role altogether with the ones of its target genes have been extensively studied. ERα and ERα-regulated genes represent the main targets in clinical approaches aimed to control hormonally responsive BC .The discovery of estrogen receptor-β (ERβ) dates back to 1996 when this molecule was cloned for the first time from rat prostate . ERβ hasThe exact role of ERβ in BC is controversial. Indeed, both proliferative and anti-proliferative ERβ roles have been described ,10. ThisSeveral coherent findings showed that ERβ expression decreases in precancerous and cancerous breast lesions ,13,14,15Differences observed in the role of ERβ are also potentially related to whether or not ERβ is expressed alone or co-expressed with ERα. In fact, ERβ appears to oppose ERα actions on cell proliferation by modulating the expression of many ERα regulated genes , includiSeveral reviews have generally addressed the topic of ERβ function in tumorigenesis ,27,28,29BC is one of the most common women cancers and represents a serious worldwide health problem. It can be considered as a multifaceted disease including a heterogeneous group of tumors with great variety in clinical, morphological, and molecular aspects . Indeed,TNBCs are known to possess a more aggressive behavior and worse disease-specific outcomes compared to ERα-positive BCs. Although TNBC accounts for ~15% of all BC cases, it is responsible for over 50% of the observed mortality ,42. IndeHowever, the advent of next-generation sequencing (NGS) has allowed for the dissection of TNBC biology and highlighted a number of molecular features typical of this cancer that could provide further insight into altered cellular pathways and identify novel therapeutic targets to face the disease ,49. IndeESR2 gene, consists of eight exons and codify for a protein of 530 amino acids, which is structurally similar to ERα and contains five distinct domains , encoded by the  domains  50]. Th. ThESR2 Besides, multiple ERβ isoforms  have beeIn BC, apart from ERβ1, the best-studied isoform is ERβ2/cx, which mediates proteasome-dependent degradation of ERα  and its In TNBC patients, high levels of ERβ2/cx have been associated with early tumor relapse . SimilarAnother explanation of the ambiguous data concerning ERβ role is the poor specificity of commercially available antibodies  and the Antibody tests, done with different methods, indicate the best performance for three of them. The monoclonal antibody PPZ0506, which targets the N-terminal region and recognizes all 5 ERβ isoforms , displayThe results concerning the frequently used monoclonal antibody 14C8, instead, are controversial; Andersson et al. , showed ERβ, similarly to ERα, can modulate gene expression in a ligand-independent manner  or upon To date, the prevalent treatment strategies of hormone-sensitive tumors are based on the use of ERα-targeting drugs. Based on the mechanisms of action exerted, the ER ligands can be grouped into two main categories : the oneAs regards SERDs, ICI 164,384 and ICI 182,780, also known as fulvestrant, are the most diffuse non-selective anti-estrogens , proposeRecently, most of the efforts have been focused on the identification of new selective ERβ agonists and many synthetic and natural molecules proved to be highly effective for BC prevention and treatment .One of the first synthetic ERβ selective ligands reported to have a high affinity for ERβ is the 2,3-bis(4-hydroyphenyl)-propionitrile, better known as DPN. Different studies reported the use of DPN as ERβ agonist in TNBC, however, the final effects of its application are discordant; Austin et al. reported that DPN is able to activate ERβ and increase TNBC cell migration ; on the More recently, several synthetic ligands such as LY700307, WAY200070, and 8β-VE2 have shown a high selectivity for ERβ in different experimental models and promising effects on proliferation, inhibition, and invasiveness suppression in BC, including TNBC ,84,85,86Among the ERβ agonists, the phytoestrogens provide a unique therapeutic opportunity to target ERβ . Two of Finally, S-Equol, a soy isoflavone metabolite, displayed a higher ERβ selectivity when compared to the other plant-derived ligands . InteresInterestingly, it has been also reported that ERβ can inhibit the pro-proliferative role of ERα also via heterodimerization with this ER subtype. Coriano et al.  using a Generally, the expression levels of ERβ decrease during breast carcinogenesis ,11,15; fRegarding TNBCs, Honma et al. have evaluated the clinical importance of ERβ in a cohort of 50 patients with TNBCs by IHC and associated ERβ1 positivity with significantly higher DFS and OS rate at 5 and 10 years . MoreoveUncontrolled cell proliferation is a crucial step in carcinogenesis. Genetic mutations and/or epigenetic modifications can lead to abnormal activation of signaling pathways that promote cell growth and inhibit apoptosis, resulting in cell cycle deregulation .In ERα positive BCs, previous studies have suggested that ERβ could exert its oncosuppressive role targeting cell division. Indeed, in breast cell lines and tumors, its expression has been inversely correlated with that of genes promoting cell cycle ,105, wheInterestingly, anti-proliferative effects of ERβ have also been reported in ERα negative BCs, including TNBC. In the TNBC cell line MDA-MB-468, Shanle et al. reported that ERβ1 exogenous expression inhibits cell growth, arresting cell cycle at G1 phase, blocks cell colony formation and reduces tumor size in mice xenografts . FurtherThe ligand-mediated anti-proliferative effects of ERβ1 have been also demonstrated by Reese et al. in other two TNBC cell lines, MDA-MB-231 and Hs578T , where eRecently, Alexandrova et al.  have alsA work from 2011 , has alsTaken together, these evidences suggest that ERβ exerts an oncosuppressive role in TNBC by targeting cell cycle genes and slowing down the cell proliferation rate.TNBCs are notoriously aggressive and prone to metastasize ,46, but Different preclinical studies have demonstrated that ERβ selective targeting with agonists could be an alternative therapeutic strategy to treat TNBC . For exaReese and colleagues, using ChIP-Seq experiments and microarray analysis, identified a gene group that is differentially regulated by ERβ after E2 or LY500307 treatment. The authors demonstrated that, in TNBC, the ligand-induced activation of ERβ generates a potent anticancer effect by the inhibition of TGF-β/SMAD pathway, which normally promotes the processes of invasiveness, cell migration, and metastasis formation .Indeed ERβ, through the recognition of specific ERE induces the synthesis of a secreted protein family, known as cystatins, that directly blocks TGFβ signaling and suppresses metastatic phenotypes, both in vitro and in vivo .Additional molecular signaling pathways seem to be involved in the invasiveness of TNBC and the development of metastasis. It has been demonstrated that almost 80% of TNBC harbor mutations in p53 gene, a well-known oncosuppressor that regulates cell cycle and, hence, inhibits tumor proliferation. The majority of these mutations generally affect the DNA-binding domain of p53 proteins that, as a consequence, lose the tumor suppressor activity and acquire a new oncogenic function. Starting from this observation, Bado et al. demonstrated that the inhibition of p53 mutant proteins is one of the mechanisms employed by ERβ to block epithelial to mesenchymal transition (EMT) and to reduce cell-invasion in TNBC .Moreover, a recent work reported that ERβ could inhibit EMT also by destabilizing the epidermal growth factor receptor (EGF-R), an oncogene expressed in basal-like cancers. These findings indicate that ERβ increases the expression of E-cadherins, a group of adhesion molecules that prevent cancer cell migration, via up-regulation of miR-200a/200b/429 and the consequent repression of ZEB1 and SIP1 , two traAltogether, these findings suggest new promising strategies to reduce the risk of metastasis in TNBC treatment, which involve the targeting of ERβ. Interestingly, starting from May 2019, the use of E2 in treating TNBC patients ERβ positive has been approved for clinical trials .Accumulation of unfolded proteins in endoplasmic reticulum (EnR), a cellular condition known as EnR stress, is observed in many cancers in response to the limited capability of tumor microenvironment to satisfy the growing request for nutrients and oxygen by cancer cells . To restOne of UPR signaling branches is regulated by inositol-requiring enzyme 1α (IRE1α), an EnR transmembrane protein that contains functional kinase and RNase domains. In normal conditions, IRE1α is bound to binding immunoglobulin protein (BIP) that prevents its dimerization and dissociates in response to EnR stress  128]. W. W128]. highCD24low population of cancer stem cells [There are several evidences indicating that IRE1α-XBP1 UPR signaling branch plays a role in TNBC tumorigenicity, one of which is the correlation between high XBP1s expression level and poor prognosis observed in TNBC patients . Anotherem cells , known tem cells ,136,137.em cells ,139, whiem cells .ERβ involvement in the TNBC cell sensibilization to EnR stress-stimulating therapy was suggested basing on the enhanced apoptosis observed in ERβ1-expressing TNBC cells treated with these drugs . InvestiEnergy metabolism represents a fundamental molecular process in cancer cells and a better understanding of mechanisms, underlying its regulation, can provide new therapeutic strategies to selectively eliminate cancer cells by targeting their unique metabolism.Adenosine triphosphate (ATP) production in normal cells relies primarily on a process called oxidative phosphorylation (OXPHOS), that occurs in the mitochondria and is regulated by mitochondrial DNA (mtDNA)-encoded genes . This waAnalysis of metabolic phenotype of TNBC tissues revealed that, similarly to genomic heterogeneity, TNBCs are characterized by high metabolic heterogeneity ,149. TheERβ localization in mitochondria has been described for the first time by Yang et al.  in 2004,Mechanisms of mitochondrial ERβ action has been extensively studied in hormone-responsive BC, where it was demonstrated that treatment with E2 enhances ERβ mitochondrial localization in a concentration- and time-dependent manner . Moreove2+, reactive oxygen, and ATP production were observed in the presence of mitoERβ [Moreover, the specific evaluation of ERβ mitochondrial role in TNBC cells has demonstrated that its mitochondria-targeted (mitoERβ) expression is able to inhibit proliferation, cell cycle progression, and reduce colony and spheroid formation . These e mitoERβ . In our  mitoERβ , GRP75 wCholesterol biosynthesis is another metabolic process, whose reprogramming favors cancer cells adaptation to environmental conditions and sustain an increased proliferation rate ,161. ChoDeregulation of cholesterol biosynthesis occurs by multiple processes including cholesterol import, synthesis, export, metabolism, and esterification ,162. SteAnalysis of cholesterol metabolic pathway activity in BC revealed that TNBC tumors are characterized by a hyper-activated cholesterol biosynthesis and efflux compared to the majority of ERα-positive BC tumors ,169. RecRecently, Alexandrova et al.  describeRecent researches have revealed that a group of TNBC expresses the AR, allowing the definition of a luminal androgen receptor (LAR) subtype ,173. MorTNBC is an extremely aggressive BC subtype that lacks well-defined therapeutic targets and for this reason, has limited treatment options, which is prompting researchers to study the molecular mechanisms that drive TNBC onset and progression. One of the suggested targets for TNBC treatment is ERβ, but to date, its role in cancer progression is still debated because of several reasons, the main of which are poor specificity of available ERβ- recognizing antibodies and existence of several receptor isoforms  that exeIn this review, we have highlighted the ERβ involvement in the different molecular pathways composing the complex network of signaling processes, regulating TNBC onset and progression.The most interesting mechanisms of ERβ action in TNBC are linked to the regulation of cellular metabolic programs, which takes place through ERβ-mediated downregulation of UPR in the presence of EnR stress , inductiAltogether, these data indicate that TNBC patients may potentially benefit from administration of ERβ-targeted therapy singularly or in combination with chemotherapeutical drugs , anti-an


Epigenome-wide association studies (EWAS), which seek the association between epigenetic marks and an outcome or exposure, involve multiple hypothesis testing. False discovery rate (FDR) control has been widely used for multiple testing correction. However, traditional FDR control methods do not use auxiliary covariates, and they could be less powerful if the covariates could inform the likelihood of the null hypothesis. Recently, many covariate-adaptive FDR control methods have been developed, but application of these methods to EWAS data has not yet been explored. It is not clear whether these methods can significantly improve detection power, and if so, which covariates are more relevant for EWAS data.In this study, we evaluate the performance of five covariate-adaptive FDR control methods with EWAS-related covariates using simulated as well as real EWAS datasets. We develop an omnibus test to assess the informativeness of the covariates. We find that statistical covariates are generally more informative than biological covariates, and the covariates of methylation mean and variance are almost universally informative. In contrast, the informativeness of biological covariates depends on specific datasets. We show that the independent hypothesis weighting (IHW) and covariate adaptive multiple testing (CAMT) method are overall more powerful, especially for sparse signals, and could improve the detection power by a median of 25% and 68% on real datasets, compared to the ST procedure. We further validate the findings in various biological contexts.Covariate-adaptive FDR control methods with informative covariates can significantly increase the detection power for EWAS. For sparse signals, IHW and CAMT are recommended. DNA methylation, as a major form of epigenetic modifications, plays a vital role in various biological processes including cell differentiation –3, genomq value procedure (ST) [With the advance of high-throughput genomic technologies, DNA methylation can now be interrogated at the genome scale. Although the gold standard for DNA methylation measurement remains bisulfite sequencing , DNA meture (ST)  are the p values. Therefore, BH and ST procedures may not be optimal when we have additional information about the hypotheses in terms of their null probability (the probability for the null hypothesis to be true) or statistical power. For EWAS data, besides the association p values, we have plenty of auxiliary covariates, which could be informative of the null probability or statistical power of the CpG-specific hypotheses. For example, differentially methylated CpG positions (DMPs) have been found to be enriched in specific genomic regions, such as promoters [Both BH and ST procedures do not differentiate hypotheses, that is, they assume that each hypothesis is equally likely to be true or false, and their rejection rule is based solely on the romoters , 31, CpGromoters , shores romoters , a speciromoters , and DNaromoters , 34. In romoters . Other sromoters , 36. Thep value thresholding (AdaPT) [Recently, there has been a surge in the development of statistical methodologies for FDR control procedures accommodating covariates with the aim to improve the detection power while still maintaining the target FDR level. Such covariate-adaptive FDR control methods include weighted FDR , the con (AdaPT) , and cov (AdaPT) . Althoug (AdaPT) , but appIn this study, we compared five covariate-adaptive FDR control methods using real data-based simulations and investigated the performance of 14 CpG-related covariates on 61 EWAS datasets. The contribution of the paper is thus threefold: (1) we developed a powerful statistical test for detecting and selecting informative covariates, (2) we identified the most robust and powerful covariate-adaptive FDR control methods for EWAS data, and (3) we recognized the most relevant covariates for EWAS data.SmartSVA) [p values  methylation datasets whose platforms were Infinium Human Methylation 450K BeadChip. The sample size of EWAS datasets ranged from 100 to 689, with an average of 211. Details about the data source and analyzed phenotypes can be found in the additional file   to captumartSVA)  and batcmartSVA) . The conmartSVA)  of the ap values under the null. Meanwhile, in order to increase the detection power, the covariate should be informative of the prior null probability or statistical power of the underlying hypotheses. For EWAS data, we investigated 14 potential covariates, which were assumed to possess the aforementioned properties. These covariates can be classified as statistical covariates  and biological/technical covariates . Statistical covariates are related to the statistical properties of the methylation data. We investigated mean of the Beta-value (“mean”), standard deviation of the Beta-value (“sd.b”) or M-value (“sd.m”), median absolute deviation of the Beta-value , measure of unimodality of the Beta-value (“dip”), inverse precision parameter of the Beta-value (“precision”) [Covariate-adaptive FDR control methods require selection of appropriate covariate(s) for the method to work. To be statistically valid, the covariate should be independent of the cision”) , sign ofp value and the covariate. Such dependency is usually explored by various diagnostic plots such as scatter plot and stratified p value histogram [p value and the covariate  for these 14 covariates to assess their informativeness  was the most informative, followed by the covariates measuring the methylation variance . For these covariates, the majority of datasets achieved significant omnibus test p values (p < 0.05). Additional file p values were enriched in small and medium Beta-values, respectively. Variance on the methylation Beta-value (“sd.b”) was more informative than that on the M-value (“sd.m”). The direction of the effect (“direction”) had mixed results, with 46.3% (25/54) of the datasets being significant. The DIP statistic (“dip”), on the other hand, was the least informative; only 12 out of 61 datasets had significant p values. For the three datasets with replicates, where we could calculate ICC values, we also evaluated the informativeness of the ICC measure (“icc.b” and “icc.m”). One dataset achieved a very low omnibus test p value (p < 0.001) while the other two were not significant, possibly due to few replicates available and an extremely sparse signal. In contrast, the biological/technical covariates were less “universal”: they were informative only on specific datasets. Overall, the relation to the CpG island (“cpg.loc”) was the most informative, followed by the position in the reference gene (“refgene.pos”) and probe type (“probe.type”). For chromosome location (“chr”) and DNase I hypersensitive site (“dhs”), they were only significant in 4 and 10 datasets, respectively.We next applied the omnibus test to the aforementioned 14 covariates based on the CpG association p values under the null [p values had an approximate uniform distribution across the strata of the covariate. Additional file A statistically valid covariate needs to be independent of the the null . We verithe null : the disMost of the existing covariate-adaptive FDR methods rely on the independence assumption, i.e., the hypotheses are not correlated. However, for EWAS data, the methylation levels of neighboring CpGs are usually correlated, violating the independence assumption. It is not clear whether these methods can still control FDR at the target level while retaining the high statistical power for EWAS data. We thus use simulation, where we know the ground truth, to study the performance of the methods. To mimic the correlation structure of EWAS data, we used a real EWAS dataset  as a temWe first studied the performance under random signal setting, where the DMPs were randomly distributed. We observed that all the methods controlled the false discovery proportion close to or under the target level (5%) across settings Fig. a. As expHowever, when the signal was sparse and the covariate was less informative, AdaPT was significantly less powerful than other methods. In contrast, FDRreg and BL performed slightly better than or similar to ST across settings while the performance of IHW varied according to the underlying signal density. IHW was less powerful than FDRreg and BL when the signal was dense, and even slightly less powerful than ST when the covariate was not very informative. As the signal became sparser, IHW was more powerful than FDRreg and BL. We also simulated correlated signals, where the DMPs tended to cluster together. Nevertheless, the pattern remained similar, indicating the robustness of these methods to correlated EWAS signals  while FDRreg and AdaPT were computationally the most intensive , a stronger correlation was observed for the covariate “mean” . Among statistical covariates, “mean,” “sd.b,” “mad,” and “precision” outperformed others  [α = 0.05) to the association p values based on the full dataset. We then compared the percentage of these fDMPs detected by CAMT with different covariates to BH/ST at different sample sizes . Significant differences in detection power were observed starting from n = 40, and peaked at n = 70. As the sample size increased continuously, the power differences became smaller and reached almost 100% for all methods at n = 100. Therefore, covariate-adaptive FDR methods could be particularly helpful for a moderately powered study . Notably, covariates “mean,” “mad,” and “sd.b” outperformed other covariates for this dataset. Similar results can be found for IHW, where we observed a similar trend  at a smaller sample size. To achieve this end, we down-sampled a large dataset EWAS51 (isorder)  and perfzes Fig. . None ofn = 583) derived from two independent studies based on purified blood cell types as described in [To see whether the covariate-adaptive FDR methods have a better power to detect age-associated DMPs (denoted as “aDMPs”), we defined a list of “gold standard” aDMPs  using CD19+ B cells and CD4+ T cells, respectively [z-score transformed p value from EWAS28 as the covariate to adjust the p value of EWAS29 to see whether we can improve the detection power of a less-powered study. We first verified the assumption of independence under the null using stratified histograms , and it appeared to be more informative than the previously studied covariates for all the covariate-adaptive methods , which plays an important role in SLE and serves as a therapeutic target [p value from a related EWAS as the covariate in FDR control serves as a convenient way to integrate previous knowledge to enrich signal and is a promising approach to an underpowered study.When there are published EWAS data from a similar disease or the same disease but from a different tissue or cell type, we may want to use the published data to improve the detection power of a new EWAS since we expect signal sharing across datasets. Covariate-adaptive FDR methods provide a convenient way to exploit previous data by using the association ectively . The sigods Fig. a. Applictes Fig. b. Remarktes Fig. b. Accordc target . Althougq value procedure [p values directly, we simulated original methylation data, upon which p values were calculated. In this way, we preserved the typical correlation structure among p values observed in real EWAS data, and thus the evaluation of false discovery rate control was more rigorous. In addition, we propose an omnibus test for assessing the informativeness of the covariates, investigate the performance of various EWAS-relevant covariates, and benchmark the methods on a large number of real EWAS datasets.In this study, we explored the use of start-of-the-art covariate-adaptive FDR control methods in multiple testing correction for epigenome-wide association testing. We also studied the performance of various EWAS-related covariates based on a large collection of EWAS data. We show that all these covariate-adaptive methods could control the false discovery rate at the target level and were substantially more powerful than traditional FDR control methods such as Benjamini-Hochberg’s step-up procedure  and Storrocedure , once inrocedure , which sFor the 61 real EWAS datasets, the most informative covariate could improve the detection power by a median of 68% and 25% for CAMT and IHW, the two best performing methods, compared to the ST procedure. Remarkably, for 9 EWAS datasets, where BH/ST procedure did not make any discovery, CAMT and IHW were able to make positive findings in four and one of the datasets using informative covariates, respectively. We further demonstrated with concrete examples that the additional DMPs recovered were biologically meaningful and could help reveal more biological insights. Given the enormous cost associated with epigenome-wide profiling, using covariate-adaptive multiple testing could potentially rescue an underpowered study, reduce the discovery cost, and ultimately shorten the bench to bedside cycle time.Although the investigated covariate-adaptive FDR control methods all maintained the target FDR level, their power varied tremendously depending on the covariate informativeness and signal density. CAMT and AdaPT were most powerful when the signal density was high or the covariate was strongly informative, suggesting their strong ability to utilizing the underlying information. However, when the signal became sparser and the covariate less informative, AdaPT suffered from a great power loss, which was consistent with the observation in . Althougp value histograms [p value and the covariate accounting for potential nonlinearity and signal sparsity. We show that the omnibus test p value correlated well with the power improvement for various covariate-adaptive methods. However, the optimal omnibus p value cutoff to achieve significant power gain differed by method and simulation studies may help determine the best cutoff.All the covariate-adaptive FDR control methods could lose power if the covariate was not informative. Thus, assessment of the informativeness of the covariate was recommended before applying a covariate-adaptive method. The traditional way of assessment relied on statistical graphics such as scatter plots or stratified stograms . Such anp value is independent of the covariate under the null. This assumption is usually assessed empirically by stratified histograms: for large p values , the distribution should appear uniform across strata. A variant of the omnibus test could be used to formally assess the assumption of independence under the null. Specifically, the omnibus test can be focused on the large p values  and tailor the p value cutoff points to be more equally spaced . We show by additional simulations  and not significant in the suggested variant .The validity of these covariate-adaptive methods depends on the assumption that the For EWAS data, we investigated 14 potential useful covariates. We found that statistical covariates are more informative than biological/technical covariates, as the former are more directly related to statistical power. In addition, statistical covariates could also be informative of the prior null probability. For example, the variance of the feature is considered to be a good indicator of the null probability  since anp value from the same or similar disease/phenotype as a covariate, and we demonstrated a substantial power gain. Using p value as a covariate provides a convenient way to conduct integrative analysis without the need for sophisticated statistical modeling or access to the raw data. Other possible covariates, which are not explored here, include the detection p value, existence of SNP in the probe, possible cross-hybridization [Besides the traditional covariates, we also explored the use of dization  and probdization  from an dization , an incrFinally, as several covariates may be informative for a particular dataset, finding the optimal combination of them will be an interesting topic. Except for IHW, all the covariate-adaptive methods can accommodate multiple covariates. Coupling the omnibus test and these methods may be a feasible solution.Covariate-adaptive FDR control methods can significantly increase the detection power for EWAS using informative covariates. The choice of the optimal method depends on the underlying signal density and the informativeness of the covariate. The informativeness of the covariate can be assessed based on the proposed omnibus test. For a dense signal, CAMT and AdaPT are the most powerful; for a sparse signal, CAMT and IHW are most promising.minfi::read.metharray.exp (version 1.26.2) [p values. Otherwise, we obtained the data from the soft file on GEO via GEOquery::getGEO (version 2.48.0) [IlluminaHumanMethylation450kanno.ilmn12.hg19 (version 0.6.0) [We collected 58 methylation datasets from GEO and split them into 61 EWAS datasets according to the phenotype, tissue source, and cell type under the following criteria: (1) the platform is Infinium Human Methylation 450K BeadChip; (2) sample size is no less than 100. Note that the majority of the 61 EWAS datasets are for different outcomes with some for the same outcome such as smoking. For datasets with raw IDAT files (the raw intensity data from the beadchip) available, R function  1.26.2)  was used 2.48.0) . The annn 0.6.0) .preprocessQuantile [minfi package. Samples with mean detection p value greater than or equal to 0.01 were removed, and probes with median detection p value across all samples less than or equal to 0.01 were filtered. Probes designed for sex chromosomes or autosomal probes co-hybridizing to sex chromosomes (cross-reactive) [The normalization of raw data was carried out by Quantile  functioneactive)  were alsisva (version 1.9) [SmartSVA (version 0.1.3) [cpg.assoc from R package CpGassoc (version 2.60) [p values were then corrected for multiple testing by various FDR control methods. Traditional FDR control methods Benjamini-Hochberg’s step-up procedure (BH) and Storey’s q value procedure (ST) were performed using the R package stats (version 3.5.3) [qvalue (version 2.12.0) [IHW (version 1.8.0) [CAMT (version 1.0) [swfdr (version 1.6.0) [adaptMT (version 1.0.0) [FDRreg (version 0.1) [FDRreg, theoretical null type was used to achieve more robust performance). See details in Table Before performing multiple testing correction, surrogate variable analysis was conducted using R package ion 1.9)  and Smarn 0.1.3)  to captuon 2.60)  and condon 2.60)  (logit.tn 3.5.3)  and qval 2.12.0) , respectn 1.8.0) , CAMT (vion 1.0) , swfdr (n 1.6.0) , adaptMTn 1.0.0) , and FDRion 0.1)  with defp value from differential methylation analysis and the covariate. Due to potential sparsity and nonlinearity of the association signal, rank-based tests  may not be powerful since the vast majority of the p values are not expected to be associated with the covariate under the null. We thus develop an omnibus test, which zooms into the low p value region and addresses nonlinearity by categorizing the covariate if it is continuous. Denote p1, p2, …, pm the p values for m CpGs and x1, x2, …, xm the values for covariate X. For a given p value cutoff pi)( (≤pi). We next consider two scenarios:X is categorical. To quantify the association between Pi)( and Xj)( and Xj) in real data analysis.For both simulated and real datasets, we use n1 = n2 = 80), based on which we added differential signals. For demonstration purpose, we only used CpGs from the chromosome 13 . To study the impact of signal density, signal strength, and informativeness of the covariate, we simulated 27 (3 × 3 × 3) settings with a varying degree of signal density , signal strength , and covariate informativeness . We first simulated “random signals,” where the differential CpGs were randomly distributed along the chromosome. Specifically, we first generated the covariate xi~N, i = 1, …, m. Given xi, we generated πi0, the probability of being the null for the ith hypothesis, asTo preserve the correlation structure observed in real data, we based our simulation on a real EWAS dataset . We drewη0 ∈ {3.5, 2.5, 1.5}, representing sparse, medium, and dense signals , and c ∈ {0, 1, 1.5}, representing a non-informative, moderately informative, and strongly informative covariate, respectively. With πi0, we generated the differential status Hi~Bernoulli(1 − πi0). If a CpG was differential, i.e., Hi = 1, we added a methylation difference fi ∈ {0.27, 0.33, 0.45}  to samples from one group, representing a weak, moderate, and strong effect. Linear regression was then performed to generate the association p values, which were further analyzed by various covariate-adaptive FDR procedures, along with the covariate. We also simulated “correlated signals,” where the differential CpGs were clustered on the chromosome. To achieve this end, we generated xi from a autoregressive model .where we set m = 10,000 hypotheses and investigated both categorical and continuous covariates. For a categorical covariate, we randomly sampled xi ∈ {1, 2, 3, 4, 5}, i = 1, …, m.. For a continuous covariate, we generated xi~N, i = 1, …, m. Next, we used the same aforementioned simulation strategy on standardized xi  to generate Hi, where η0 ∈ {3.5, 2.5, 1.5}, representing sparse, medium, and dense signals, and c ∈ {0, 0.25, 0.375}, representing no dependence, weak dependence, and moderate dependence on the covariate, respectively. To simulate a nonlinear dependency for a continuous covariate, we replaced xi with πi0. Next, we generated the z-score zi~N, if Hi = 0, and zi~N if Hi = 1. We finally generated the p value based on 1 − Φ(zi), where Φ is the c.d.f of the standard normal.We simulated ns in R package splines, version 3.5.0) [We investigated 14 covariates “mean,” “sd.b,” “sd.m,” “mad,” “dip,” “precision,” “direction,” “icc.b,” “icc.m,” “refgene.pos,” “cpg.loc,” “chr,” “dhs,” and “probe.type.” The definition, calculation, and representation of these covariates could be found in Table n 3.5.0) . Besidesp value below 0.05 based on the full sample size were defined as fDMPs. Since EWAS51 was of binary phenotype, we subsampled each phenotype to a sample size 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100. At each sample size, 100 replications were performed.We used EWAS51  to perform down-sampling analysis. CpGs with Bonferroni-corrected gometh function of R package missMethyl (version 1.16.0) [GO and KEGG enrichment analysis was done by  1.16.0) . All CpGtidyverse (version 1.2.1) [ggplot2 (version 3.1.1) [ggforce (version 0.3.0) [ComplexHeatmap (version 2.1.0) [UpSetR (version 1.4.0) [ggpubr (version 0.1.7) [cowplot (version 1.0.0) [For data cleaning and transformation, R package n 1.2.1)  was appln 3.1.1) , ggforcen 0.3.0) , Complexn 2.1.0) , UpSetR n 1.4.0) , ggpubr n 0.1.7) , and cown 1.0.0) . ClusterAdditional file 1:Table S1. Description of the Datasets.Additional file 2:Table S2. Informativeness statistics for the 14 covariates based on the omnibus test.Additional file 3:Table S3. Percentage of BH-significant DMPs recovered by IHW with different covariates.Additional file 4:Table S4A. Reproducing the smoking EWAS results on EWAS20. Table S4B. Additional smoking-associated DMPs detected by CAMT.Additional file 5:Table S5. Top 20 GO Terms based on ST Results.Additional file 6:Supplementary figures.Additional file 7:Review history.


AbstractValdiviaserrata in Colombia as Temnocephalaivandarioisp. nov. The most distinctive characters of the new species are in the cirrus and the epidermal ‘excretory’ syncytial plates. In the present study, the terminology to describe the cirrus of species of Temnocephala is updated. Comparison between the shape of the cirrus of the temnocephalans associated with trichodactylid crabs is also provided.A new species of temnocephalan is described from the branchial chambers of  Temnocephala Blanchard, 1849 includes 37 species of symbiotic freshwater rhabdocoels from the Neotropics, which are associated with a large variety of hosts including chelonians (3), mollusks (6), insects (7) and crustaceans (21)  . At presbouvieri , Phallanadispar  and StreValdiviaserrata White, 1847 (crab) were manually collected from the Reserva Natural Tanimboca, Leticia, Amazonas , Colombia. The specimens were transported alive to the Programa de Estudio y Control de Enfermedades Tropicales (PECET) laboratory and identified using a decapod key  and 70% ethanol for morphological identification by light microscopy; or fixed in 2.5% glutaraldehyde for observation by Scanning Electron Microscopy (SEM). The temnocephalans were identified by focusing on the morphology of the reproductive complex, the shape of the epidermal excretory syncytial plates (DLSPs) and the deposit areas of the eggs in the host. Terminology to describe the cirrus of the temnocephalans was updated from SEM to determine the shape and position of the egg filament, the fracture plane of the eggshell, the shape of the DLSPs and the relative position of the excretory pore. Measurements were in micrometres (µm) unless otherwise indicated; ranges were determined followed by the arithmetic mean, the standard deviation and the number of specimens measured for a given character . Photomicrographs of the temnocephalans were taken with a Nikon Alphaphot YS-2 microscope. Drawings were made using a drawing tube Nikon 1.25X. Line drawings and photographic images were prepared using Inkscape 0.92. The SEM preparations were examined with a Hitachi S-4800 SEM at the Servicio de Microscopía, SCSIE, Universitat de València, Spain. Type specimens were deposited in the Colección Colombiana de Helmintos (CCH.116), Universidad de Antioquia, Medellín, Colombia.Eleven specimens of apod key . SpecimePageBreakPlatyhelminthes Minot, 1876Phylum Rhabdocoela Ehrenberg, 1831Order Dalytyphloplanida Willems, Wallberg, Jondelius, Littlewood, Backeljau, Schockaert & Artois, 2006Suborder Neotyphloplanida Willems, Wallberg, Jondelius, Littlewood, Backeljau, Schockaert & Artois, 2006Infraorder Limnotyphloplanida Van Steenkiste, Tessens, Willems, Backeljau, Jondelius & Artois, 2013Parvorder Temnocephalida Blanchard, 1849Section Temnocephaloidea Baer, 1953Superfamily Temnocephalidae Monticelli, 1899Family Taxon classificationAnimaliaRhabdocoelaTemnocephalidaeAB659D89-132A-5A69-AF77-FB117A431E6Ahttp://zoobank.org/E7284E9B-D311-46C3-B5CF-26CA544B15EFValdiviaserrata White, 1847 , Colombia.Kilómetro 11, Reserva Natural Tanimboca, Leticia, Amazonas (Holotype: CCH.116 (159); Paratypes: CCH.116 (160).SEM, 5 unhatched eggs observed by SEM.10 whole mounted specimens; 5 stained in Meyer’s paracarmine; 5 stained in Borax carmine; 6 dissected cirrus; 2 samples observed by External characteristics. Body (without tentacles) 1.36–2.26 mm (1.75 ± 0.25) long by 1.18–1.56 mm (1.36 ± 0.11) wide; adhesive disk ventral, subterminal 280–520 (370 ± 82) long by 320–520 (400 ± 40) wide ; excretory pore “subcentral” in the DSLP, displaced towards the internal limit  long by 210–310 (248 ± 32) wide; pharynx 330–620 (417 ± 56) long by 450–620 (511 ± 52) wide; intestine saccular, without septations , long by 60–145  wide. Vagina elongated with strong muscular wall, connects to the genital atrium dorsally, 75–180  long by 16–30  wide with a widening of the distal portion; proximal vaginal sphincter symmetrical 16–34  and distal vaginal sphincter symmetrical   long by 172–212  wide. Vitellarium arborescent and thin  long by 302–331  wide; filament small, subapical or apical  Figs , 4A, B. hin Fig. . Eggs 55cal Fig. ; pedunclcal Fig. . Eggs decal Fig. .PageBreakPageBreakMale. Two pairs of testes, medium-sized, usually rounded, slightly oblique, anterior testes 180–310 (231 ± 34) long by 120–320 (220 ± 48) wide; posterior testes 200–400 (260 ± 60) long by 110–360 (254 ± 65) wide  long, with maximum width 15–22  at level of the distal portion  in the distal portion of the introvert, without spines or ridges in the inner wall of the introvert . Temnocephalaivandarioi sp. nov. is the third species described from Colombia, and therefore V.serrata is registered as a new trichodactylid host for neotropical temnocephalans. Valdiviaserrata is widely distributed throughout the Orinoco and Amazon River basins in Venezuela, the islands of Trinidad and Tobago, the Guianas, Colombia, Brazil, Peru and Bolivia  for each species. The morphology of the cirrus constitutes one of the few characters used and is the most valuable taxonomic character for species identification . In the , accordiabs Fig.  are compThe cirrus is defined as the entire sclerotised male copulatory organ comprised of a ‘shaft’ (rigid, tubular region tapering distally; Fig. The shape of the shaft may be described as a ‘funnel’, ‘goblet’, or ‘cone’. Funnel- or goblet-shaped shafts have a wide proximal region which tapers rapidly into a narrow tubular distal region . The cirThe introvert shape can be described as ‘cylindrical’, ‘cone’; ‘scoop’, or ‘goblet’. Scoop- or goblet-shaped introvert have a wide middle region, which tapers into a narrow distal region. In addition, the introvert may be ‘unarmed’, armed with ‘grooves’ in the proximal limit of the introvert, or armed with ‘spines’, ‘sclerites’, and ‘ridges’ in the inner wall of the introvert. The distal opening of the introvert may be at right angles with respect to the proximal limit of the introvert i.e. ‘not oblique’, ‘oblique’, or ‘very oblique’ (modified from The morphology of the cirrus is necessary for species identification and should be clearly described based on the terminology proposed in the present study. This new terminology can be applied to species of neotropical temnocephalans described to date.


During Ca2+ transients, Ca2+-binding to troponin C shifts the “on–off” equilibrium of the thin filament state toward the “on” sate, promoting actomyosin interaction. Likewise, recent studies have revealed that the thin filament state is under the influence of temperature; viz., an increase in temperature increases active force production. In this short review, we discuss the effects of temperature on the contractile performance of mammalian striated muscle at/around body temperature, focusing especially on the temperature-dependent shift of the “on–off” equilibrium of the thin filament state.In skeletal and cardiac muscles, contraction is triggered by an increase in the intracellular Ca Under physiological conditions, striated muscle generates force and heat. Skeletal muscle plays a critical role in maintaining body temperature which increases during exercise. Human body temperature is maintained at ∼37 ± 1°C throughout the day . In huma2+ from the sarcoplasmic reticulum (SR) via ryanodine receptors; however, in cardiac muscle, it is the Ca2+ entry from the extracellular fluid through voltage-dependent L-type Ca2+ channels that triggers the Ca2+ release, a mechanism known as Ca2+-induced Ca2+ release (2+concentration ([Ca2+]i) promotes Ca2+ binding to troponin C (TnC) on thin filaments (2+]i is maintained relatively low (∼10–6 M), even at the peak of systole  and tropomyosin (Tm), which form a complex on actin filaments. At rest, the Tn–Tm complex prevents/weakens actomyosin interaction. At this “off” state, the carboxyl-terminal domain of TnI strongly binds to actin, and Tm blocks myosin binding to actin and/or force production of bound myosin. When [Ca2+]i is increased, Ca2+-bound TnC interacts with TnI, and the carboxyl-terminal domain of TnI is dissociated from actin. The Tn conformational changes result in displacement of Tm on actin, which subsequently induces myosin binding to actin and force generation   is widely used to express Ca2+ sensitivity; an increase in the pCa50 value indicates an increase in Ca2+ sensitivity and vice versa. Ca2+ sensitivity is influenced by various factors, such as the intracellular concentrations of Mg2+   : see eceptors , causingnotropy)   , hence, 2+ transients is likely to underlie the inhibited active force production.In contrast, an increase in temperature to ∼40–42°C has been reported to decrease end-systolic pressure in canine  and rat Physiologists have realized for nearly a century that despite being under resting conditions, the warming of muscles increases active force, known as “heat contraction” or “heat rigor”. For instance, 2+-independent manner, and instead, it is regulated at the sarcomere level. Indeed, intracellular Ca2+ imaging with fluo-4 revealed little or no increase in [Ca2+]i upon infrared laser irradiation, and heating-induced contraction was blocked by the myosin II inhibitor blebbistatin. A similar phenomenon was observed in C2C12 myotubes (from mouse) when temperature was increased from 36.5 to 41.5°C using gold nanoshells in combination with near-infrared laser irradiation  induces transient and reversible shortening in isolated intact rat ventricular myocytes . In thisadiation . These s fashion .2+ sensitivity is increased with increasing temperature above 37°C   see , and intat ∼50°C .in vitro motility assay, recent studies confirmed heating-induced activation of thin filaments by measuring the sliding velocity of reconstituted thin filaments. 2+ (+EGTA). We performed a rapid-heating experiment using infrared laser irradiation and found that thin filaments that had been reconstituted with bovine cTn and human Tm exhibited sliding movements at >∼35°C in the absence of Ca2+  and Tm, modulate CaM or HCM . A generutations . Becauset manner , local hIt is worthwhile noting that in previous studies discussed, thus far, different mammals were used that have different body temperatures cf. , thus, f2+]i. The Ca2+-dependent structural changes of thin filaments allow for myosin binding to actin and thereby facilitate active force production. Cooling increases the contractility of striated muscle via Ca2+-dependent activation: first, a rapid decrease in temperature triggers a release of Ca2+ from the SR, and second, long-term cooling increases the amplitude as well as the period of intracellular Ca2+ transients. Contrary to these cooling effects, heating increases myofibrillar active force (and ATPase activity) and Ca2+ sensitivity; the latter is coupled with an increase in the affinity of TnC for Ca2+. Moreover, heating induces structural changes of thin filaments , thereby shifting the “on–off” equilibrium of the thin filament state toward the “on” state at a given [Ca2+]i (Ca2+-independent activation). The characteristics of heating-induced, Ca2+-independent activation may be useful to augment the heart’s contractility in patients with DCM in future clinical settings.In striated muscle, sarcolemmal depolarization causes an increase in [CaSIs, KO, and NF wrote the first version of the manuscript. SS, FK-S, and S’Is contributed comments and suggestions. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Maternal hypertension is a common and serious condition associated with increased maternal and foetal morbidity and mortality, with early detection and management improving outcomes.Blood pressure (BP) changes of pre-eclampsia are defined after 20 gestational weeks, while haemodynamic changes can be detected at 5–11 weeks using a specialised non-invasive Doppler stroke volume (SV) monitor. Thus, advanced haemodynamic monitoring allows for physiologically precise identification of circulatory abnormalities, and implementation of appropriate therapy within the first trimester. We measured the oscillometric BP and advanced haemodynamics (USCOM 1A) of 3 unselected women with singleton pregnancies, consecutively listed for therapeutic induction for maternal hypertension at 32–41 weeks gestational age. While the BP’s of the patients varied, it was the haemodynamics, particularly SV, cardiac output, systemic vascular resistance, Smith Madigan Inotropy Index, and oxygen deliver, that identified differing patterns of circulatory dysfunction, therapeutic objectives, and predicted post-partum complications of the mother and child.First trimester screening of maternal haemodynamics may allow for earlier detection of circulatory derangements, selection of patient precise interventions, and improved maternal-foetal outcomes. Hypertension in pregnancy is a common and serious condition associated with significantly increased maternal and foetal morbidity and mortality, as well as increased cost of care –5. WhileThe heart and vessels function to deliver oxygen and substrates to the cells and are regulated by the autonomic nervous system (ANS) and its sensors, predominantly baroreceptors, which act to preserve BP. As Guyton demonstrated, BP is the product of cardiac and vascular function with its interdependence described by the equation .\documenmentclass2pt{minim2 . Th•. Th6•].2, plus 20 additional advanced haemodynamic parameters [2. The USCOM 1A has been proven to be reliable, reproducible and more sensitive than bio-impedance [−5, at the end of pregnancy are associated with higher risks of foetal distress and maternal complications [The non-invasive USCOM 1A Doppler monitor  has recently been validated for advanced point of care monitoring of haemodynamics in pregnancy –24. The rameters . SMII isrameters  and is ampedance •, 29, anmpedance ••, with mpedance ••. Estabmpedance ••, 19•• mpedance ••, whileications  and pretications . Tiralonications ••. Whileications •, 7•, thications •, 32, 33Maternal hypertension is often treated with induction and Caesarean section •, which The monitoring of maternal haemodynamics has demonstrated promising results for the early identification and patient precise targeting of treatment of maternal hypertension , 36, 37•Contemporaneously measured BP and Doppler SV haemodynamic examinations were performed on three randomly selected women with singleton pregnancies at 32 to 41 weeks gestational age following informed consent. Participants were consecutively listed for therapeutic induction for maternal hypertension in the 24 h following testing at a maternal tertiary referral centre in a Chinese provincial hospital. Maternal haemodynamics were measured using the USCOM 1A MATERNAL haemodynamic monitor  and BP measures acquired using routine brachial oscillometry , with all measures acquired 5 min following the subjects placed in the supine resting position. The BP and USCOM haemodynamic measures were collated and compared. Normal values for pregnant haemodynamics were referenced from Vinayagam et al. ••. GradiNormal adaptive circulation2) with a normal BP of 119/65, MAP 83, and one prior normal pregnancy with mild late term hypertension, for induction under anaesthesia at 41 weeksThirty-two-year-old  presenting with mild hypertension, 137/92 and MAP 107 mmHg and a history of one prior pregnancy complicated by mild hypertension. For planned induction at 36 weeks.Normal vaginal delivery of a 2780-g baby at 32 gestational weeks with an Apgar score of 8. Mother and baby recovered quickly with an overnight observational ICU stay.2, with a mildly decreased SVR  with moderately elevated SV, CO, HR, SMII and DOSevere vasogenic hypertension with severely impaired cardiac function.2) with 3 prior pregnancy’s complicated by moderate hypertension, presented at 32 weeks gestational age with severe hypertension, BP 165/95 (MAP = 118) and increasing oedema and leg pitting. The patient was scheduled for a therapeutic induction at 32 weeks gestational age.A 33-year-old obese mother . Additionally, there was severe impairment of the SV , CO (4 v 6.9 L/min), SMII (1.1 v 2.0 W/m2) and, importantly, DO2 was approximately 60% of normal (594 v 900 ml/m2) (Fig. Summary—Severe vasogenic hypertension, with a severely elevated SVR (2332 v normal 1100 dyne s cmPhysiologically guided therapy would have targeted reducing the SVR with a vasodilator, and an inotrope to stimulate the LV function.Outcome—The baby, after Caesarean birth, had a depressed 5 point APGAR score, which recovered over the first hour, while the mother required 3 days of ICU care. It is probable that this significant haemodynamic dysfunction would have been expressed in the first trimester and would have been detected by haemodynamic monitoring. This may have led to improved physiologically targeted management and a more stable haemodynamic course through pregnancy and improved outcome.2 for normal and hypertensive subjects (Figs. 2, the therapeutic targets, correlated with MAP, the current method of defining and guiding therapy in maternal hypertension.Table ts Figs.  and 10, 2 and SMII and their impact on maternal-foetal outcomes. This improved understanding may allow for early implementation of patient precise therapy.These three case studies of third trimester hypertensive pregnant subjects demonstrate the variability and unpredictability of maternal outcomes by BP, while abnormal haemodynamics more closely predicted outcomes Table . AdvanceThis observational study demonstrates the simple principles and application of advanced haemodynamic monitoring in identifying and managing maternal hypertension but is limited in its scope. Study of more subjects, randomised across a range of BPs, is required to better define the potential benefits of this physiologically guided approach. Further study of the temporal evolution of normal and pathological maternal haemodynamics may identify new personalised therapeutic approaches to its management and establish the utility of a routine first trimester scan.2 may lead to an earlier and more accurate diagnosis, more precise therapy and improved outcomes in maternal hypertension.Advanced haemodynamic monitoring in pregnancy provides identification of circulatory pathophysiology at 5 to 11 weeks, and defines quantitative therapeutic targets not identified by BP alone. First trimester screening of SV, CO, SVR, SMII and DO