Continuous invariant-based maps visualize for the first time all two-dimensional lattices extracted from hundreds of thousands of known crystal structures in the Cambridge Structural Database. This paper develops geographic style maps containing two-dimensional lattices in all known periodic crystals parameterized by recent complete invariants. Motivated by rigid crystal structures, lattices are considered up to rigid motion and uniform scaling. The resulting space of two-dimensional lattices is a square with identified edges or a punctured sphere. The new continuous maps show all Bravais classes as low-dimensional subspaces, visualize hundreds of thousands of lattices of real crystal structures from the Cambridge Structural Database, and motivate the development of continuous and invariant-based crystallography. Data sets of this size require finer classifications than by 230 crystallographic groups.Crystallography traditionally splits crystals into only finitely many classes, for instance by their space-group types. These discrete symmetry-based classifications were suitable for distinguishing highly symmetric crystals manually or simply by eye. Nowadays crystals are simulated and synthesized on an industrial scale. The Cambridge Structural Database (CSD) contains nearly 1.2 million existing crystal structures , v2(t) =  in Fig. 2t varies continuously in . Since the initial basis v1 = , v2(0) =  and final basis v1 = , v2(1) =  define identical lattices, this continuous family of lattices is a closed loop in the space of all lattices. For v1 = , v2(t) =  is reduced by Definition 2.1Consider the family of lattices with the basis t passes through v1 = , v1 = , v1 = , v0(t) =  will be a new reduced basis for v1, v1, v1 to the longer vector.When equivalence relation (denoted by ∼) satisfies the axioms: (i) reflexivity: any lattice Λ is equivalent to itself, so Λ ∼ Λ; (ii) symmetry: if Λ ∼ Λ′, then Λ′ ∼ Λ; (iii) transitivity: if Λ ∼ Λ′ and Λ′ ∼ Λ′′, then Λ ∼ Λ′′.One way to call lattices identical  is to ignore deviations of lattice parameters up to a certain threshold. An equivalence gives rise to a justified classification only if this equivalence classes: the class [Λ] consists of all lattices equivalent to Λ, since if Λ is equivalent to Λ′, which is equivalent to Λ′′, all three lattices are in the same class. Past equivalences in the work of Lima-de-Faria et al. I, such as a numerical vector, is called an isometry invariant of a lattice I takes the same value on all isometric lattices: if Λ ≅ Λ′ are isometric then I(Λ) = I(Λ′), so I has no false negatives. An isometry invariant I is called complete (or injective) if the converse also holds: if I(Λ) = I(Λ′) then Λ ≅ Λ′, so I distinguishes all non-isometric lattices. Hence a complete invariant I has neither false negatives nor false positives  = I(Λ′). For instance, infinitely many non-isometric lattices have the same primitive cell area.In a fixed coordinate system, the basis vectors are not isometry invariants as they change under rotation, but the primitive cell area is preserved by isometry. If an invariant Continuous invariants, which change only slightly under small perturbations of the underlying object, are even better. The dependence of pseudosymmetry on thresholds discussed by Zwart et al.  of any lattice d satisfying all necessary axioms and the new continuity condition below:Find a complete isometry invariant d = 0 if and only if Λ ≅ Λ′ are isometric;(i) First axiom: d = d for any lattices (ii) Symmetry axiom: d + d ≥ d for any lattices (iii) Triangle axiom: C such that, for any lattices d ≤ Cɛ.(iv) Lipschitz continuity: there is a constant bThis paper applies a solution of Problem 1.2 from Kurlin 2022b to visua2.et al., 2016v3 whose length becomes infinite. For vectors v1 =  and v2 =  in v1, v2 is defined as Crystallography traditionally uses a conventional cell to uniquely represent any periodic crystal (see Hahn Definition 2.1 (reduced cell)U are called reduced (non-acute) if |v1| ≤ |v2| and v1| ≤ |v2| and special condition for rigid motion is: if |v1| = |v2| then v1v2 ≥ 0.For a lattice up to isometry, a basis and its cell a × b, but two ‘potentially reduced’ bases v1 =  and v2 = , which are not related by rigid motion for 0 < a < b. Definition 2.1v1 =  and v2 = . So The new conditions for rigid motion did not appear in the work of de Wolff 2016 because d taking the same non-zero value  for any non-isometric lattices et al.  consisting of all points V(Λ) uniquely determines Λ up to rotations, almost any tiny perturbation of a rectangular lattice Λ converts the rectangular domain V(Λ) into a hexagon. Hence all combinatorial invariants (numbers of vertices or edges) of V(Λ) are discontinuous, similarly in higher dimensions.Another way to represent a lattice  Λ Fig. 5. Though ababHowever, comparing Voronoi domains as geometric shapes by optimal rotation v1, …, vn in superbasev0, v1, …, vn from Conway & Sloane  has a reduced (non-acute) form with 0 < q11 ≤ q22 and −q11 ≤ 2q12 ≤ 0, which is equivalent to reducing a basis up to isometry.Definition 2.2oane 1992, which woane 1992 and Delav1 = , Q = 9x2 − 6xy + 5y2 satisfying the reduction conditions above. So quadratic forms do not distinguish mirror images (enantiomorphs). Hence the new conditions for the rigid motion were needed in Definition 2.1The bases v0 + v1 + v2 = 0 changes p12, p01, p02 by the same permutation of indices. For example, swapping v1, v2 is equivalent to swapping p01, p02.Motivated by the non-homogeneity of the metric tensor , Delaunay 1937 proposedoane 1992  and root invariants RI, RIo]B = {v0, v1, v2} be any obtuse superbase of a lattice v0, v1, v2 have different lengths and no right angles, and hence can be ordered so that |v1| < |v2| < |v0|. Let sign(Λ) be the sign of v1, v2. The root invariant RI(Λ) is the triple of the root products i, j ∈ {0, 1, 2}. The oriented root invariant RIo(Λ) is RI(Λ) with sign(Λ) as a superscript, which we skip if sign(Λ) = 0.Let rij = rji. If |v1| < |v2| < |v0|, then r12 < r01 < r02. If some vi, vj have equal lengths, then rik = rjk for k ≠ i, j. Writing RI(Λ) =  means that |v1| ≤ |v2| ≤ |v0| for a suitable indexing of obtuse superbase vectors v0, v1, v2.We assume that bB because an obtuse superbase of Λ is unique up to isometry, also up to rigid motion for non-rectangular lattices. This uniqueness was missed by Conway & Sloane size σ(Λ) = r12 + r01 + r02 and projects RI(Λ) to x, y) is the projected invariant. The oriented invariant PIo(Λ) is obtained by adding the superscript sign(Λ).The root invariants of all lattices o(Λ) with sign(Λ) live in a union of two quotient triangles QT+ ∪ QT−. These triangles should be glued along the common subspace of mirror-symmetric lattices .All oriented projected invariants PIExample 3.3 (subspaces of Bravais classes in QT).a × a has RI(Λ4) =  ∈ TC projected by TP to (tp) The square lattice 6 with a minimum inter-point distance a has the root invariant 6) =  =  ∈ QT For a lattice a) the invariant RI(Λ) uniquely identifies Λ up to isometry,(b) the invariant RIo(Λ) uniquely identifies Λ up to rigid motion,(c) the invariant PI(Λ) uniquely identifies Λ up to isometry and uniform scaling,(d) the invariant PIo(Λ) uniquely identifies Λ up to rigid motion and uniform scaling.(a): any lattices only if) direction means that if Λ ≅ Λ′ are isometric, then RI(Λ) = RI(Λ′), so RI(Λ) is an isometry invariant taking the same value on all isometric lattices. The second (if) direction means that if RI(Λ) = RI(Λ′), then Λ ≅ Λ′ are isometric.Each part in Theorem 3.44.v2, v3}, {v1, v3}, {v1, v2} of given basis vectors of Λ. So the CSD provides a huge collection of 2.6 million two-dimensional lattices, which our reduction approach maps to the triangle QT in under 1 h on a standard laptop.For any periodic crystal structure from the CSD, which has full geometric data of its lattice Fig. 9v1, v2 whose angle is perturbed from 90° by less than 0.03°. The CSD has only 399 such lattices and The gap of about two pixels near the horizontal edge in Fig. 9a ≃ 3.5 Å arising from 386 near-identical primitive monoclinic crystal structures of α-oxalic acid dihydrate. This molecule was used as a benchmark for the calculation of electron densities since its crystallographic properties were thoroughly documented by Stevens & Coppens  among CSD lattices.Hexagonal and square lattices are characterized by the inter-point distance 5.v0, v1, v2 permutes the three root products accordingly: QΛ = q11x2 + 2q12xy + q22y2 representing Λ is not homogeneous in the above sense. Taking square roots gives the quadratic invariant QI(Λ) =  = a).In comparison with other complete invariants, RI(Λ) has the advantage of homogeneity so that any permutation σ of (indices of) superbase vectors bet al.  of all mirror-symmetric lattices Λ.Since the quotient square QS = QTP+ (pole), the centre of the circle inscribed into QT+ because the rays from P+ to the vertices of QT+ equally bisect the angles 90°, 45°, 45°. The incentre of QT+ has the coordinates , where v1 ≃ , v2 ≃  inversely designed by Kurlin [2022b2)].We can choose any internal point of the quotient triangle QT as the north pole. The most natural choice is the incentre Definition 5.1 a) The spherical map SM sends the incentre P+ of QT to the north pole of the hemisphere HS+ and the boundary ∂QT to the equator of HS+  is the angle from the equatorial plane EP of S2 to the radius-vector to the point SM[PIo(Λ)] ∈ S2 in the upwards direction. Let v(Λ) be the orthogonal projection of this radius-vector to EP. Define the Greenwich point as P+ and . This G represents all centred rectangular lattices with a conventional unit cell 2a × 2b whose ratio Greenwich meridian is the great circle on the sphere S2 passing through the point SM(G) in the equator E. The longitude μ(Λ) ∈  above. and is proved in Appendix AFor lattices with PI(Λ) in the straight-line segment between the excluded vertex  and the incentre Proposition 5.2 (formulae for SM)x, y) ∈ QT, if y − t)90°.For any lattice The longitude of the lattice Λ isThe latitude isP± ∈ QT± have ψ = 0 and φ = ±90°, respectively, μ is undefined.The incentres Example 5.3 (prominent lattices). Any mirror-symmetric lattice E of S2 and has φ(Λ) = 0 by (2)4 with PI(Λ4) =  has 6 with PI(Λ4) =  has Greenwich lattice ΛG with PI(ΛG) = G = P+ whose pixel contains 230 lattices in Fig. 10The north pole represents the incentre The heat maps show a hexagonal ‘ridge’ along the meridional arc at μ = −45° in Figs. 146.et al.  of a lattice Λ is complete, this RI(Λ) could be said to represent the DNA of Λ. Even better than the real DNA, any two-dimensional lattice can be explicitly built up from RI(Λ) [see Kurlin duplicates of T2-γ and two (near-)duplicates of T2-β reported in our past work (Pulido et al., 2017et al. (2022Working towards a complete materials genome, Widdowson  al. 2022, section al. 2022 predicte


Although tremendous efforts have beenmade to improve the membrane’s ion selectivity, increasingthe ion selectivity without sacrificing the proton conductivity isstill a challenging issue. In this work, instead of focusing on enhancingthe membranes’ ion selectivity, we develop an efficient valenceregulation strategy to suppress the capacity decay caused by the crossoverof V2+ in VRFBs. Despite the discharge capacity of theVRFB with the elevated average valence electrolytes (V3.68+) being slightly lower than that with commercial electrolytes (V3.50+) in the first 35 cycles, the accumulated discharge capacityin 400 cycles is improved by 52.33%. Moreover, this method is efficient,is easy to scale up, and provides deep insights into the capacitydecay mechanism of VRFBs.Nafion series membranesare widely used in vanadium redox flowbatteries (VRFBs). However, the poor ion selectivity of the membranesto vanadium ions, especially for V 3.50+), the accumulated dischargecapacity in 400 cycles of a VRFB withthe elevated average valence electrolytes (V3.68+) is improvedby 52.33%.Compared withthe commercial electrolytes (V In VRFBs, the redox couples of VO2+/VO2+ and V2+/V3+ are adopted as the active species on the positiveand negative sides, separated by an ion exchange membrane. Ideally,the V3+ (V2+) content in the anolyte shouldbe equal to that of VO2+ (VO2+) in the catholyte to ensure that theelectrolytes have the highest available capacity. However, the electrolytestate gradually deteriorates with cycling  due to the membrane’sunideal ion selectivity, resulting in rapid capacity decay.A vanadium redox flow battery(VRFB) is one of the most promising large-scale energy storage technologiesdue to its high safety, long lifespan, easy scalability, and flexibledesign, which makes it viable for large-scale energy storage systems in the next 10–15 years.2+ has a much higher diffusionrate across the Nafion series membranes than other vanadium ions (V2+ > VO2+ > VO2+ >V3+),3 which results in thenet flux of V2+ transport from the negative to the positiveside.5 Meanwhile, side reactions occur after V2+ transportsto the positive side:6Nafion series membranes are the most widely used proton exchangemembranes in VRFBs. However, V2+ reacts with VO2+ directly  of the electrolyte on the positive side are finally increased,but the VO2+ is scanty on the positive side and continues to intensify with cycling.7 Oppositely, the negative electrolyte volume decreasesduring the cycling, but some V2+ cannot be used due tothe scanty VO2+, leading to the unavailable V2+ accumulating on the negativeside. Such a volume imbalance, valence imbalance, and concentrationimbalance of the electrolytes on the positive and negative sides significantlyreduce the capacity of VRFBs.Overall,no matter whether Vdirectly 1 or reacith VO2+ 2 and 3, 8 polybenzimidazole(PBI),9 and polypropylene (PP)10 with Nafion or modified the Nafion membraneswith microporous materials such as zeolites11 and oriented graphene oxide (GO)12 toenhance the membranes’ ion selectivity. However, the delaminationof such composite structures13 is usuallyinevitable, and the tradeoff between ion selectivity and proton conductivityremains challenging.14Numerous works have focused onimproving the ion selectivity ofNafion series membranes in VRFBs to suppress the capacity decay. Someresearchers composited Teflon,15 or redividing the electrolytes after remixingthe positive and negative electrolytes16 has been applied to partially restore the capacity of VRFBs. However,these methods can only partially balance the contents and valencestates of electrolytes on the positive and negative sides but areineffective in reducing the crossover and capacity decay rate.Moreover,introducing excess electrolytes on the negative side18 adopting a mixed acid system,20 and increasing the operating temperature (with some organic/inorganicadditives),24 such research is mainly focused on improving the energy efficiencyof VRFBs. There has been no research on improving the capacity retentionof VRFBs by optimizing the configuration of electrolytes.Even though the electrolyte utilization of VRFBs can be enhancedto some extent by optimizing the sulfuric acid concentration,2+ accumulationon the negative side during cycling by generating surplus VO2+ at the first charge/dischargecycle. Although elevating the average valence of electrolytes sacrificesthe capacity of VRFBs in the first 35 cycles to a certain extent,the elimination of unavailable V2+ during cycling can greatlydecrease the crossover of V2+ and significantly improvethe cycling performance. Notably, the accumulated discharge capacityof VRFBs with V3.68+ in 400 cycles is improved by 52.33%from 256.97 to 391.43 Ah in comparison to that with commercial balancevalence electrolyte (V3.50+).Inthis work, by exploring the capacity decay mechanisms of VRFBswith Nafion membranes in depth, we developed an electrolyte with elevatedaverage valence to eliminate the unavailable V3.50+) and the concentration of differentvanadium ions varying with cycling was tested (detailed informationis shown in the Supporting Information),as shown in 2+ and V3+, which can reflect the total contents of vanadiumions after fully discharging and oxidizing V2+ with air.As wasmentioned above, the concentration of different vanadium ions varieswith cycling due to their different diffusion rates across the Nafionmembranes. Herein, the discharge capacity of VRFBs with commercialelectrolytes . The viscositydifferences increase with cycling and finally arrive at a dynamicbalance between diffusion and convection, where the discharge capacityof VRFBs almost keeps a stable state25 (atabout 200 cycles). Besides, the voltage efficiency (VE) of VRFBs decreasesslightly with the cycle number . This indicates that the decay of VE and discharge capacity canbe ascribed to the increasing concentration loss caused by electrolyteimbalance during cycling.26 In other words,it can be deduced that the capacity decay of VRFBs is exclusivelycaused by the crossover of vanadium ions.ve sides S5. The v2+ is the only vanadium ionon the positive side after being fully discharged, while both V2+ and V3+ exist in the negative electrolytes inthe VRFB after crossover. However, it is difficult to detect the remainderof V2+ after fully discharging because the absorbance signalof V2+ (562 nm) is weak and close to the peak of V3+ (610 nm) in the UV spectrum.25 Therefore, the generation of unavailable V2+ with cyclingis confirmed by testing the concentration variation of V3+ before and after oxidation in the air. As shown in 3+ increases profoundly after oxidizing the electrolytes with air,while the concentration of VO2+ almost remains the same, the volume and content of the positive electrolytesincrease butVO2+ is scantydue to the crossover of V2+ after long-term cycling. Meanwhile,the volume and content of negative electrolytes decrease, but theunavailable V2+ accumulates with cycling  contributeto the crossover separately. Moreover, the concentration and volumeof the electrolyte increase on the positive side and decrease on thenegative side with cycling7 due to themuch higher diffusion rate across the Nafion series membranes of V2+ in comparison to other vanadium ions.3 The electrolyte change during VRFB cycling results in asurplus of V2+ on the negative side.5 Therefore,we used the net flux of V2+ (the amount of vanadium ion(V2+/ V3+) crossover from the negative to thepositive sides subtracted by the amount of vanadium ion (VO2+/VO2+) crossover from the positive to the negativesides) to depict the effects of vanadium ion crossover on the ionevolution in the charge/discharge process for brevity.Nevertheless, theoxidation of Vx (0< x < 0.5 due to the two consumptions of VO2+) is the net fluxratio of V2+ transport from the negative to the positiveside by crossover, and y is the oxidation ratio ofV2+ with air. Thus, the oxidation ratio of V2+ with air (y) effects on the capacity of VRFBs underdifferent crossover conditions (x) can be deduced, as shown in Herein,the initial capacity of VRFB is defined as 1; 2+ with air decreases the capacityof VRFBs at the initial stage (area A), which is consistent with thecurrent popular perception.9 However, no researchhas yet revealed that the oxidation of V2+ with air canrestore the capacity to different degrees in areas B–D of 2+ with air on the capacityof VRFB, VRFBs with the commercial electrolyte (V3.50+)are tested with and without air oxidation during cycling, as shownin 2+ oxidized by air during 400 cycles. Ox-4 denotes a VRFB with4 times of air oxidation of V2+ at the 50th, 100th, 200th,and 300th cycles during cycling . Noticeably, a gradientof decreasing current density (312.5–200–125–62.5–50–18.75mA cm–2) was applied before oxidizing the negativeelectrolyte with air to ensure the accumulated unavailable V2+ is oxidized completely without excess oxidation , the discharge capacity of VRFBs will keep stableduring cycles 200–300 if we oxidize the unavailable V2+ with air in a timely and continual manner. Therefore, we testedthe VRFB (swnoted Ox-C) performance with continual air oxidation ofnegative electrolyte during cycling ), as shown in 3+ in the anolyte.On the otherhand, since the capacity decay in cycles 200–300is caused by a valence unbalance , and V3.50+ is the balance valence of the fresh commercial electrolyte. 3.50+, while that of Ox-0-remixed is very close to V3.50+.Moreover, elevating the average valence of electrolytes will decreasethe maximum capacity of a VRFB,28 and thisis also reflected in the VRFBs with the remixed electrolytes of Ox-4and Ox-C, as shown in Infact, the average valence of electrolytes will increase to someextent after oxidizing the negative electrolytes with air, which isvalidated by the UV results after remixing the electrolytes on thepositive and negative sides, as shown in Figure S8.2+ resultsin the lack of VO2+ on the positive side and the accumulation of unavailableV2+ on the negative side, which significantly aggravatesthe capacity decay. The VRFB with an elevated valence electrolytegenerates surplus VO2+ after a charge/discharge cycle  arriving earlier than that of Ox-0 and the freshelectrolyte  is compared with the commercial balancedvalence electrolyte (denoted V3.50+) in Inspired by the stable performance of Ox-4-remixed and Ox-C-remixed,we first proposed a strategy to suppress the capacity decay of VRFBsby elevating the average valence of electrolytes. We prepared a freshelectrolyte with an elevated average valence according to the remixedelectrolyte of Ox-4 after 400 cycles due to its good performance 4c. Speci3.68+ issuccessfully suppressed compared with that of V3.50+, andit presents the same trend as that with Ox-4-remixed . Meanwhile,benefiting from the smaller concentration loss, the VE of VRFB withV3.68+ is also remarkably improved over that of V3.50+ , implying that elevating the valenceof electrolytes can effectively suppress the vanadium ion crossoverand improve capacity retention. Besides, the discharge capacity andvoltage efficiency of the VRFB coupled with V3.68+ presenta larger fluctuation frequency with cycling than that of the VRFBcoupled with V3.50+, but they display a similar fluctuationamplitude. The reason for the fluctuation is the varying room temperature,which changes regularly with time in our laboratory. However, thedischarge capacity of the VRFB coupled with V3.50+ is muchsmaller than that with V3.68+ after 200 cycles . This work provides deep insights into the capacitydecay mechanism of VRFBs and develops an efficient and easily scaled-upelectrolyte design method for suppressing the capacity decay of VRFBs,showing great potential for engineering applications in the near future.We investigated the unavailable VSupporting Information.No unexpected or unusually high safety hazards were encountered.Details of experiments and the deduction process of crossover effectson vanadium ion concentration changes are described in


Background. Fibrillatory Wave Amplitude (FWA) has been described as a non-invasive marker of atrial fibrillation (AF) complexity, and it predicts catheter ablation outcome. However, the actual determinants of FWA remain incompletely understood. Objective. To assess the respective implications of anatomical atrial substrate and AF spectral characteristics for FWA. Methods. Persistent AF patients undergoing radiofrequency catheter ablation were included. FWA was measured on 1-min ECG by TQ concatenation in Lead I, V1, V2, and V5 at baseline and immediately before AF termination. FWA evolution during ablation was compared to that of AF dominant frequency (DF) measured by Independent Component Analysis on 12-lead ECG. FWA was compared to the extent of endocardial low-voltage areas , to the surface of healthy left atrial tissue, and to P-wave amplitude in sinus rhythm. The predictive value of FWA for AF recurrence during follow-up was assessed. Results. We included 29 patients. FWA remained stable along ablation procedure with comparable values at baseline and before AF termination , whereas DF significantly decreased . FWA was higher in LVA-I than in LVA-II, -III, and -IV in Lead I and V5 (p = 0.02 and p = 0.01). FWA in V5 was strongly correlated with the surface of healthy left atrial tissue . FWA showed moderate to strong correlation to P-wave amplitude in all leads. Finally, FWA did not predict AF recurrence after a follow-up of 23.3 ± 9.8 months. Conclusions. These findings suggest that FWA is unrelated to AF complexity but is mainly determined by the amount of viable atrial myocytes. Therefore, FWA should only be referred as a marker of atrial tissue pathology. Atrial fibrillation (AF) is the most frequently encountered cardiac arrhythmia and is responsible for significant morbidity and mortality . Based oDespite important improvements since its initial description, catheter ablation of AF remains complex and time-consuming, and appropriate selection of candidates for ablation is critical. The time and frequency domains analyses of the fibrillatory wave on the surface ECG has gained increasing interest over the past decades for predicting catheter ablation outcomes, which can help in determining the most appropriate treatment for patients with persistent and long-standing persistent AF .Fibrillatory wave amplitude (FWA) was one of the first ECG-derived parameters to be evaluated for assessing AF complexity, and previous studies found that patients with lower FWA presented more frequent ablation failures, because of a presumably higher degree of AF complexity ,4,5,6. FIn this study, we sought to evaluate the links of FWA with anatomical atrial substrate and AF complexity by assessing the evolution of FWA during radiofrequency catheter ablation of persistent and long-standing persistent AF. We also compare FWA to AF dominant frequency, to the amount of left atrial (LA) low voltage areas and of healthy LA tissue, and to P-wave voltage after sinus rhythm recovery.Consecutive patients undergoing first time radiofrequency ablation for persistent or long-standing persistent symptomatic drug-refractory AF at Pasteur University Hospital, Nice, France, were included. The study was approved by the institutional review board, all patients gave written informed consent, all methods were performed in accordance with the relevant guidelines and regulations, and research was performed in accordance with the declaration of Helsinki.Procedures were performed under general anesthesia. Transesophageal echocardiography (TEE) was performed before venous puncture for ruling out left atrial appendage (LAA) thrombus. A decapolar catheter was positioned in the coronary sinus (CS), through a transfemoral venous access under vascular echography guidance. A single or double transseptal puncture was performed under TEE guidance. A bolus of unfractionated heparin was administered immediately after the transseptal puncture and infusion was titrated to maintain activated clotting time >300 s for the duration of the procedure. Procedures were performed without interruption of vitamin K antagonists with International Normalized Ratio < 3, and new oral anticoagulants were interrupted the day before the procedure.High-density electroanatomical mapping >1000 points) of the entire LA was performed with the Carto system V6 or V7 , using the Pentaray catheter (Biosense Webster Inc.) inserted transseptally via a non-steerable sheath . The Thermocool SmartTouch SF ablation catheter (Biosense Webster Inc.) was inserted through the transseptal puncture in another SL0 sheath. Detailed mapping of electrical activation of the LA  was performed, with visual annotation of zones of spatiotemporal electrogram dispersion (STD)  and of c00 points2) were assessed using high-density electroanatomical mapping with the Carto system, after excluding the inside portion of PVs and the mitral valve. The surface of healthy atrial tissue was determined by subtracting the surface of LVA to the total LA surface.LA volume (mL) and LA surface  disconnection, ablation at STD and CFAE sites, and block across the lines (if performed). Roof lines, anterior mitral lines, or lateral mitral lines were performed in cases of left atrial substrate in the area (presence of STD or CFAEs). There was no recommendation regarding the order of the treated LA areas, and ablation could be initiated either by PV isolation or by left atrial substrate ablation. Each area was strictly ablated in a sequential manner, for allowing assessment of the effect of each area ablation on AF complexity parameters. LA areas were defined as: left PVs ostia, right PVs ostia, roof, anterior, septal, floor, lateral isthmus, and LAA base.High-power short duration irrigated radiofrequency was delivered using a power of 50 W and with an ablation index target of 350 arbitrary units (AU) on the posterior wall of the LA, and 420 AU elsewhere. If CS ablation was performed, radiofrequency power was limited to 25–30 W.AF termination during ablation was defined as sinus rhythm resumption or its change to a stable atrial tachyarrhythmia. Nevertheless, this was not a procedural endpoint, and operators ended the procedures after PV isolation, complete ablation of the annotated STD and CFAE sites, and, when appropriate, block across the lines. Associated atrial tachyarrhythmia ablation was performed, and the critical isthmus or focal origin was specifically targeted up to sinus rhythm resumption and reconfirmation of PV isolation before catheter withdrawal. In cases without AF or atrial tachyarrhythmia termination at the end of the procedure, an electrical cardioversion was performed .A blanking period of 1 month was chosen in this study, because early arrhythmia recurrences at 1–3 months have proven to be predictive of late arrhythmia recurrences ,13,14. AECG signals were acquired on a digital electrophysiological recording system , including 0.05 to 40 Hz band pass and 50 Hz notch filters. For every patient, a 1-min standard 12-lead ECG in AF was recorded at a sampling rate of 1000 Hz at the beginning of the procedure, after each LA area ablation, and immediately before AF termination . Additionally, a 1-min standard 12-lead ECG was recorded in sinus rhythm at the end of the procedure.A previous study designated Leads I, V1, V2, and V5 as the most discriminant to determine mid- and long-term catheter ablation outcome based on FWA , and theThe outcomes of the signal processing algorithm used to compute the FWA in each lead is illustrated in Once in sinus rhythm at the end of the procedure, P-wave amplitude was measured. Because P-wave is stable over time, its amplitude was assessed by peak-to-peak measurement.AF dominant frequency (DF) is related to the refractory period atrial myocardium cells, and thus to the degree of complexity of the disease and the probability of spontaneous cardioversion . In thisThe atrial activity source is automatically selected as the extracted component with a dominant peak in the interval [3–9] Hz, the typical AF frequency band. The percentage of signal power around the dominant peak, or spectral concentration (SC), has been shown to correlate with atrial activity extraction quality  and is ht-test as appropriate, whereas a non-parametric Mann—Whitney U-test was used when the variables did not show a normal distribution. When more than two groups were present, ANOVA analysis was performed. Proportion analysis was based on the chi-square test. Correlation between continuous variables was performed using the Pearson’s R coefficient. Statistical significance was defined by a p value < 0.05.Statistical analyses were performed using SPSS Statistics v20 software . Distribution normality of continuous variables was checked with the Kolmogorov—Smirnov test. Categorical variables were presented as percentages and continuous variables as mean and standard deviation in the case of distribution normality, or median and confidence interval otherwise. Under data normality, groups were compared by a parametric unpaired or paired 2 per patient, representing 8.9 ± 3.6% of the total LA surface.We included 29 patients . Clinical and procedural characteristics are given in p = 0.49; Lead V1 p = 0.59; Lead V2 p = 0.71; and Lead V5 p = 0.75) nor with DF using RobustICA or PCA . However, baseline DF was strongly inversely correlated with LAA cycle length .Baseline FWA was neither correlated with LAA cycle length (Lead I p = 0.535), Lead V1 , Lead V2 , and Lead V5 . These results remained unchanged when only the patients whose AF terminated during radiofrequency application were analyzed .FWA remained stable along the ablation procedure in all studied leads; baseline FWA was similar to FWA before AF termination in Lead I  and PCA . Spectral concentration value was higher using RobustICA than using PCA , evidencing the superior atrial activity source separation using RobustICA and suggesting the better performance of this technique for DF assessment.In contrast, DF significantly decreased during ablation using RobustICA . There was also a significant correlation with baseline FWA in Lead I and Lead V2, but with a weaker correlation coefficient . No correlation was evidenced with FWA in lead V1 (p = 0.178).There was a strong correlation between the surface of healthy LA tissue and baseline FWA in lead V5  and in Lead V5 . See The mean surface of LVA was 46 ± 31.4 cmAmongst the 29 patients, 12 (41.4%) were LVA-I (<10% of low-voltage areas compared to LA surface), 5 (17.2%) were LVA-II (10–20% of low-voltage areas), 4 (13.8%) were LVA-III (20–30% of low-voltage areas), and the remaining 8 (27.6%) were LVA-IV (>30% of low-voltage areas). p = 0.007), and there was a trend towards a correlation for Lead I (p = 0.1). For Leads V1 and V2, no link was evidenced between FWA and LVA stages . FWA was significantly higher in patients with LVA-I stage compared to LVA stage ≥II in Lead V5  and in Lead I , but not in Leads V1 and V2 . However, LVA stage was not related to DF , AF duration (p = 0.81), or LA volume (p = 0.35).FWA in Lead V5 was significantly correlated with LVA stages (p = 0.01), Lead V2 , and Lead V5 . No significant correlation was seen with Lead I (p = 0.16).After sinus rhythm resumption, P-wave amplitude was moderately correlated with the surface of healthy LA tissue in Lead V1 . P-wave amplitude after sinus rhythm resumption was significantly correlated with baseline FWA in all studied leads, with a moderate to strong correlation coefficient , as well as AF duration  and LA volume . However, baseline FWA was similar in both groups: 14.2 ± 6.5 vs. 12.4 ± 3.7 µV in Lead I (p = 0.44), 48.9 ± 22.5 vs. 44.4 ± 11.4 µV in Lead V1 (p = 0.56), 39.6 ± 14.1 vs. 39.4 ± 11.5 µV in Lead V2 (p = 0.97), and 22.3 ± 6.9 vs. 27.1 ± 10.5 µV in Lead V5 (p = 0.18). The surface of healthy LA tissue and the surface of LVA were not linked with AF termination (p = 0.109 and p = 0.324).AF termination during radiofrequency was obtained in 19/29 patients (65.5%); the remaining 10 patients underwent electrical cardioversion at the end of the procedure. Baseline DF was lower in patients for whom AF termination was reached per ablation as compared to those who underwent electrical cardioversion at the end of the procedure , a larger LA (238 (226–251) vs. 165 (141–177) ml; p = 0.0252) and a higher DF (6.53 (6.47–6.59) vs. 5.62 (5.24–6.09) Hz; p = 0.0371). However, FWA was similar in recurring AF or non-recurring AF patients in Lead I (11.4 (9.1–13.7) vs. 12.8 (11.2–16.0) µV; p = 0.5467), Lead V1 (51.4 (37.1–65.7) vs. 40.8 (36.2–52.6) µV; p = 0.3438), Lead V2 (45.7 (43.0–48.3) vs. 35.2 (30.3–43.3) µV; p = 0.7963) and Lead V5 (29.8 (25.0–34.5) vs. 22.0 (18.0–26.1) µV; p = 0.1212). The surface of healthy LA tissue and the surface of LVA were not correlated with AF recurrence (p = 0.572 and p = 0.728).After a follow-up of 23.3 ± 9.8 months, 2/29 patients (6.9%) had AF recurrence and 11/29 patients (37.9%) demonstrated left atrial flutter; 16/29 (55.2%) remained free from any arrhythmia after a single ablation procedure. Patients with recurring AF had a longer AF duration before ablation (72 (48–96) vs. 12 (6–27.3) months; In this study, we assessed FWA during persistent AF ablation and compared it to its assumed determinants: the degree of AF complexity and the amount of viable LA tissue.Our results suggest that FWA is unrelated to AF complexity. First, at baseline, FWA did not demonstrate any correlation with DF or LAA cycle length, which are established parameters of AF complexity. Second, FWA was stable in all leads along the ablation procedure, with similar values at baseline, where AF is the most complex, and immediately before AF termination or cardioversion, where AF is assumed to be the least complex. In contrast, DF demonstrated a significant decrease during the ablation procedure. Finally, FWA was neither correlated with AF termination during the ablation procedure, nor with AF recurrence during follow-up, whereas DF, LA volume, and AF duration were correlated with both.On the other hand, FWA was significantly correlated with the amount of LA low-voltage areas and with the surface of healthy LA tissue, with the best results for lead V5, which better displays LA electrical activity due to its localization on the left precordium. Additionally, FWA was correlated in all studied leads with P wave amplitude in sinus rhythm, which is fundamentally uncomplex and is directly related to atrial tissue quality.All these findings point to the fact that FWA is a marker of atrial tissue pathology, but not a direct parameter of AF complexity.Several previous studies have assessed the value of FWA for predicting AF recurrence after catheter ablation, using manual ,5 or autDuring catheter ablation of AF, a progressive organization occurs and AF complexity gradually decreases ,26. As sAs a marker of atrial tissue pathology, FWA assessment remains valuable for predicting complex ablation procedures, because AF genesis and maintenance is strongly linked with atrial fibrosis . HoweverOur group of patients is relatively small, with 29 patients included. With a larger number of patients, it is possible that a significant decrease in FWA before AF termination would have been demonstrated, especially in Lead V5. As discussed previously, this lead better displays LA electrical activity, and a decrease in FWA after ablation could be explained by the reduction of viable LA tissue.The assessment of DF was carried out by means of atrial source separation, taking into account all ECG leads simultaneously, and it is unknown whether sequential single lead analyses using the Fourier Transformation method would have given comparable results.Follow-up was performed in “real-life” settings by sequential 24-h Holter monitoring and symptoms assessment, but implantable loop recorders were not used.Finally, LVA were defined by a bipolar voltage <0.25 mV, as suggested in a previous study , but no Our study suggests that FWA is not related to AF complexity but is mainly determined by the amount of viable atrial myocytes. Therefore, FWA should only be considered as a marker of atrial tissue pathology.


The nanostructure of hybrid nanobiocomposites produced by extrusion was analyzed by X-ray diffraction and transmission electron microscopy, and the morphology was analyzed by scanning electron microscopy. In addition, selected mechanical properties and thermal properties were studied by thermogravimetric analysis, TGA, and differential scanning calorimetry, DSC. The interactions of the composite ingredients were indicated by FT IR spectroscopy. The effect of the amount of nanofiller on the properties of prepared hybrid nanobiocomposites was noted. Moreover, the non-equilibrium and equilibrium thermal parameters of nanobiocomposites were established based on their thermal history. Based on equilibrium parameters , the degree of crystallinity and the mobile and rigid amorphous fractions were estimated. The addition of Cloisite®30B and aliphatic polyurethane to the P3HB matrix caused a decrease in the degree of crystallinity in reference to the unfilled P3HB. Simultaneously, an increase in the amorphous phase contents was noted. A rigid amorphous fraction was also denoted. Thermogravimetric analysis of the nanocomposites was also carried out and showed that the thermal stability of all nanocomposites was higher than that of the unfilled P3HB. An additional 1% mass of nanofiller increased the degradation temperature of the nanocomposites by about 30 °C in reference to the unfilled P3HB. Moreover, it was found that obtained hybrid nanobiocomposites containing 10 wt.% of aliphatic polyurethane (PU400) and the smallest amount of nanofiller (1 wt.% of Cloisite®30B) showed the best mechanical properties. We observed a desirable decrease in hardness of 15%, an increase in the relative strain at break of 60% and in the impact strength of 15% of the newly prepared nanobiocomposites with respect to the unfiled P3HB. The produced hybrid nanobiocomposites combined the best features induced by the plasticizing effect of polyurethane and the formation of P3HB–montmorillonite–polyurethane (P3HB-PU-MMT) adducts, which resulted in the improvement of the thermal and mechanical properties.This paper presents an attempt to improve the properties of poly(3-hydroxybutyrate) (P3HB) using linear aliphatic polyurethane (PU400) and organomodified montmorillonite (MMT)—(Cloisite With the growing awareness in society about environmental care, the use of biodegradable plastics is now the best solution to the environmental risks posed by conventional and commonly used plastics. Biodegradable plastics are environmentally friendly, and their properties can be compared with those of conventional polymers. Many applications have been, including in food packaging and in biomedicine ,16,17,18Poly(3-hydroxybutyrate) is produced as an energy carrier from various bacteria ,23,24, wPolymer nanocomposites are currently an important direction in industrial development. The addition of even a small amount of nanofillers to a polymer leads to improvement in their mechanical, thermal, barrier, flammability, ductility, stiffness, and electrical and thermal conductivity properties, without affecting their processability ,31,32,33®30B) on the performance properties and structure of P3HB biopolymer. It was expected that P3HB-based hybrid nanobiocomposites would exhibit improved properties due to two different reinforcing mechanisms of the used modifiers.The present work investigated the effect of aliphatic linear polyurethane and organomodified montmorillonite  = 5.72; the P3HB melt flow index was 0.11 g∙(10 min)−1 (180 °C at 2.16 kg). Organically modified montmorillonite Cloisite®30BB was supplied by Southern Clay Products Inc. . Cloisite®30BB is a natural montmorillonite modified with methylbis(2-hydroxyethyl)tallowalkylammonium cations.P3HB was supplied by Biomer ; its weight average molecular mass was MHexamethylene 1,6-diisocyanate (HDI) and dibutyltin dilaurate (DBTL) were purchased from Sigma-Aldrich , and polyethylene glycol, with a molecular mass of 400 g/mol (PEG400), from Merck .w = 13.430 g/mol and the dispersity was Mw/Mn = 2.09. Linear polyurethane was synthesized by the procedure described in [The molecular weight of the PU400 determined by size exclusion chromatography (SEC) in tetrahydrofuran (THF) was Mribed in .where m is 8.7 and n is 8.6 on average.®30B. The mixture was then stirred for about 20 min at room temperature for homogenization. To homogenize the components, P3HB was introduced into a Stephan-type mixer, followed by an appropriate amount of liquid PU400 and an appropriate amount of CloisiteThe homogeneous mixture was then introduced into the hopper of a co-rotating twin-screw extruder with a working diameter of D = 25 mm and L/D = 33, operating at 300–450 rpm. The different zones of the extruder were maintained at the following temperatures during the extrusion of the composite: hopper: 22–36 °C, II zone: 120–125 °C, III zone: 135–145 °C, IV zone: 134–136 °C, V zone: 134–135 °C, VI zone: 145–148 °C, VII zone: 145–148 °C, VIII zone: 148–163 °C, IX zone: 134–150 °C, and head: 158 °C to 170 °C.During the extrusion, the volatile parts were evacuated by degassing, and the temperatures of the head of the extruder and the heating zones of the plasticizing system of the extruder were kept constant. The molten composite was cooled in a cooling bath, pelletized, and dried at 60 °C for 2 h.The unfilled P3HB and a mixture of P3HB containing 10 wt.% of PU400 (C-10-0) were also extruded in order to compare the properties of the prepared nanobiocomposites with a reference material.3), V1, V2—volumes of the KOH solution used for the titration of the blank sample and the analyzed sample, respectively, (cm3), m—mass of the sample, (g).The hydroxyl number (HN) of polyethylene glycol and the obtained polyurethane were determined according to the standard (PN-87/C-89085/13). For this purpose, 1 g of the tested compound was placed in a conical flask and the acetylating mixture, i.e., a solution of acetic anhydride in DMF, was added. The flask was equipped with a reflux condenser, and the solution was heated in a boiling water bath for one hour. After heating was completed, toluene was poured into the flask and stirred vigorously. Then, it was titrated with 1.5 M potassium hydroxide solution in the presence of phenolphthalein until a persistent pink color appeared. The hydroxyl number value was calculated from the formula:aq in the presence of methyl red until a bright red color persisted. A blank test was performed in parallel. The contents of the isocyanate groups (ICN) in % by weight were calculated using the equation:1 and V2—volumes of 0.1 M HClaq used for the titration of the blank and the sample, respectively, (cm3); m—mass of the sample, (g); 0.0042—the number of isocyanate groups equivalent to 1 cm3 of a 0.1 M HCl solution, (g).The content of the isocyanate groups (ICN) in the synthesized polyurethane was determined according to the standard (PN-EN 1242:2006). A 0.5 g sample was placed in a conical flask, dissolved in acetone, and mixed with 0.2 M acetone ammonia solution. The flask was stoppered, the solution was stirred, and then left standing for 10 min at room temperature. The excess ammonia was titrated with 0.1 M HClThe X-ray diffraction technique was used to characterize the structure of biocomposites at the nanometric scale. The measurements were carried out at room temperature with the use of a Bruker SAXS Nanostar-U X-ray diffractometer. The spectra of the samples were studied in transmission mode to verify the effect of nanoparticle orientation. A small-angle diffractometer was coupled to a filtered CuKa radiation source (1.54 Å) in a sealed tube, operating at 50 kV and 30 mA. The 2D detector (Vantec2000) was used to scan the entire sample surface; the spot beam was approx. 500 μm. The scanning range was determined by changing the distance of the sample from the detector. The resolution of the detector and the angular range allowed for measurement at the level of 2048 × 2048 pixels. The measurements were carried out from 1 to 28° over a period of 2 h.The nanostructures of the nanobiocomposites were tested by transmission electron microscopy (TEM). The nanoparticle distribution was imaged using a Tecnai TF20 X-TWIN (200 kV)—a high-resolution transmission electron microscope equipped with a field emission gun (FEG) . TEM samples were prepared with the use of a microtome.The study of the composite morphologies was carried out using a JEOL JSM-6490 LV scanning electron microscope with an accelerating voltage of 20 kV and secondary electron detection (SEI). The samples to be tested were subjected to freezing in liquid nitrogen and then broken using a blade hammer. The surfaces of the broken samples were then sputtered with a thin layer of gold with a thickness of about 10 nm using a JEOL JFC-1300 sputtering machine. Finally, the sputtered samples were placed in an electron microscope chamber, and the analysis of the material surface was conducted within several micro-areas.The infrared (FT IR) spectra of the P3HB, polyurethane, and their composites were recorded on an ALPHA FT-IR Bruker instrument in the ATR mode or in KBr pellets.on), the temperature corresponding to 50% weight loss (T50%), the temperature of the maximum decomposition rate (Tmax), and the total weight loss of the samples at 600 °C were determined.Thermogravimetric analysis (TGA) of the nanobiocomposites was conducted using a Metler Toledo TGA/DSC 3+ thermogravimetric analyzer. The composite samples were heated at a rate of 5 °C/min in the temperature range of +25 to +600 °C in an inert gas (nitrogen) atmosphere. The temperature of the onset of decomposition (Tm(onset) = 156.6 °C), and the heat of fusion (ΔHf = 28.45 J/g (3.281 kJ/mol)). In order to obtain accurate results, the heat capacity was calibrated by sapphire [All experiments by calorimetry in the temperature range from −90 °C to 195 °C were performed using a Q1000TM differential scanning calorimeter (DSC) from TA Instruments, Inc. . This calorimeter is the heat-flux type and is equipped with a mechanical refrigerator to control the heating and cooling of samples. The measurements were carried out in a nitrogen atmosphere with a constant flow rate of around 50 mL/min. The mass of samples used for measurements by DSC was about 10 mg. The experimental heat flow was measured at a heating rate of 10 °C/min after previously cooling at a rate of 10 °C/min by the standard DSC. The temperature and heat flow rate calibration in the DSC apparatus was performed using the parameters of the melting indium, the onset melting temperature ).The impact strength of the nanobiocomposite samples was performed using an Instron brand impact hammer. The impact test was carried out for bilaterally notched samples according to method A of the standard (PN-EN ISO 179-1:2010).The hardness of the obtained nanobiocomposite samples was determined by the Shore method using Bareiss Shore hardness testers: HP-A and HP-D. The measurement consisted of measuring the sample resistance of the tested composite while immersing an indenter needle with a specific shape and dimension in accordance with the standard (PN-EN ISO 868 and ASTM D2240).®30B) nanoadditive were produced using a co-rotating twin-screw extruder. Polyurethane obtained in the reaction of 1,6-hexamethylene diisocyanate and polyethylene glycol with a molar mass of 400 g/mol was used in the amount of 10 wt.%, and Cloisite®30B in the amounts of 1, 2, and 3 wt.%, . In order to obtain a reference material to compare the properties with the obtained nanobiocomposites, unfilled P3HB and a mixture of P3HB with 10 wt.% PU400 (C-10-0) were used.Hybrid nanobiocomposites based on P3HB with linear aliphatic polyurethane and modified montmorillonite (MMT)  did not show a diffraction maximum below the value of 2θ < 5°. This means that the P3HB chains penetrated between the layers of the nanoclay platelets and there was not only separation, but complete delamination. On the other hand, the introduction of 2 and 3 wt.% of Cloisite®30B resulted in a significant broadening of the null peak. In addition, a low-intensity peak was observed with a diffraction maximum at 4.64°, corresponding to the second reflection (d002). Its presence suggests the possible small share of the partially intercalated structure of the nanobiocomposites.To characterize the nanoclay structure of the prepared nanobiocomposites, the X-ray diffraction (XRD) technique was used in the range of 1° to 30°. For comparison, reference diffractograms of unfilled P3HB and CloisiteMoreover, the diffractograms of the prepared nanobiocomposites indicate an increase in the amorphous degree of the semicrystalline matrix P3HB. This is evidenced by the shape change of peaks in the range of 2Θ from 19 to 27°.®30B. P3HB chains penetrated the silicate gallery, with complete delamination (exfoliation) on single silicate nanoplatelets. However, the influence of the nanofiller concentration was not observed at the level of exfoliation. To emphasize this state, the micrograph with less magnification (200 nm) was shown for the nanobiocomposite with the highest concentration (3 wt.%) of Cloisite®30B (C-10-3).The nanobiocomposites were observed by the TEM method to identify the nanostructure features. The structure analysis of the resulting nanobiocomposites by TEM confirmed the conclusions resulting from the XRD analysis. The TEM micrographs of the nanobiocomposites are shown in The fractured surface of the so-called null unmodified polyester (P3HB), shown in The next micrograph shows the surface of the blend loaded with 10 wt.% of polyurethane based on PEG400. As can be seen, the introduction of polyurethane (PU400) as the only modifier caused an apparent disruption of the continuity of the P3HB matrix structure. The presence of crystalline domains is noticeable. The rough areas visible in the image, which are arranged in different directions, suggest the interaction of the biopolymer matrix with the PU400, resulting in the separation of the interacting P3HB chains and the formation of the mentioned domains.®30B).The following micrographs in ®30B (1 wt.%) had a noteworthy completely different structure from the other samples. Distinct crystalline domains arranged in different directions were much larger in size than in the other accompanying photos. In addition, these domains were separated by larger distances than in the other samples, suggesting a smaller interaction between them and a flexibilizing effect of the P3HB.The polyester matrix modified with 10 wt.% of PU400 and the smallest amount of Cloisite®30B caused a further decrease in the TS. The greater the addition of nanofiller, the greater the decrease in stress, and it reached about 17.5% for the nanobiocomposite containing 1 wt.% of Cloisite®30B (C-10-1) and 36.0% for the nanobiocomposite with 3 wt.% of Cloisite®30B (C-10-3). ®30B showed higher impact strength than the original sample. However, the maximum impact strength (an improvement of 15% compared to the unfilled P3HB) was shown by the composite designated as C-10-1. It should be noted that the impact strengths of the nanobiocomposites C-10-2 and C-10-3 were lower compared to the unmodified P3HB. As illustrated in The average impact strength (IS) values are shown in ®30B increased by 30 °C compared to the unfilled P3HB. However, a further increase in the amount of nanofiller made the thermal stability of the nanobiocomposite unchanged.Thermogravimetric analysis (TGA) was carried out in order to study the thermal stability of the prepared nanobiocomposites . The TGA®30B caused a slight decrease in the thermal stability of the nanobiocomposites, but the thermal stability nevertheless remained superior to that of polyester and polyester modified with 10 wt.% of PU400 (biocomposite designated C-10-0). The fastest decomposition temperatures for P3HB and its hybrid nanobiocomposites were similar . The exception was P3HB modified with PU400 alone, in which case a temperature drop of 5 °C was observed.The addition of 3 wt.% of Cloisite®30B was introduced, and it increased with its increasing content. This was related to the increase in the content of inorganic aluminosilicate in the nanobiocomposite.The decomposition residue decreased when PU400 was introduced, and it increased again when Cloisiteg, and the change in the heat capacity at Tg, ΔCp, were established based on the study of the glass transition region, and the heat of fusion, ΔHf, and the onset melting temperature, Tm, were also estimated based on the analysis of the melting region. Additionally, it can be noted that the double melting peak was maintained as with the P3HB analysis. This is probably related to the presence of crystals of different lamella thicknesses [Qualitative thermal analysis was carried out based on the heat flow rate of the P3HB and its nanobiocomposites using DSC. cknesses .®30B additive on the glass transition temperature (Tg) and melting temperature (Tm), and the variations in the heat of fusion and the heat capacity at Tg were investigated. ®30B. Based on the qualitative analysis  of the unfilled semicrystalline P3HB and its nanobiocomposites, the changes in the heat capacity at Tg and the heat of fusion with Tm were estimated. It was observed that the values of the glass transition temperatures were lower than those of the unfilled P3HB. This is proof of the plasticizing effect of Cloisite®30B on the P3HB matrix. The biggest changes in the glass transition temperature of 13.3 °C and the melting temperature of 7.7 °C were observed between the P3HB and C-10-3. The incorporation of polyurethane reduced the glass transition temperature to 0.7 °C [The influence of the Cloisiteo 0.7 °C .on-Tm (onset) = 250.0 °C–154.3 °C).Particularly interesting and useful is the substantial difference between the values of the degradation and the melting temperature, which facilitates processing and prevents the degradation of the material. It can be noted that the best thermal properties and the widest processing window expressed as the difference between the degradation temperature and the melting temperature were obtained for C-10-2 and equaled 95.7 °C (based on Tc) and the mobile (Wa) and rigid amorphous (WRAF) phases  in the range 0.11–0.56 J·°C−1·g−1. The melting endotherm of the semicrystalline C-10-1 occurred between 152 and 161 °C with a heat of fusion in the range 2.7–101.1 J·g−1. It should be noted that the changes in the heat capacity values were estimated from the qualitative thermal analysis . The red points are characteristic of a two-phase model. In turn, the black squares confirm the existence of a rigid amorphous fraction and characterize the three-phase model. The solid straight line indicates the full amorphous and full crystalline C-10-1 nanobiocomposite represented by the yellow star points. The change in the heat capacity for the fully amorphous C-10-1 was 0.58 J·°C−1·g−1, and the heat of fusion for the fully crystalline samples was 114.2 J·g−1.a = ΔCp/ΔCp100% and the degree of crystallinity using Wc = ΔHf/ΔHf100%. The degree of crystallinity (Wc) was defined as the ratio between the experimental heat of fusion (ΔHf) of the semicrystalline sample and the heat of fusion of the fully crystalline C-10-1 sample (ΔHf100%). ΔCp and ΔCp100% are the variations in the heat capacity at Tg (mobile) of the semi-crystalline and full amorphous C-10-1, respectively. For computation, a ΔCp100% value equal to 0.58 J·°C−1·g−1 and ΔHf100% = 114.2 J·g−1 were used. The green triangle point derived from the qualitative analysis of the C-10-1 is also plotted in RAF = 1 − Wa − Wc). Similar analyses for the other nanocomposites were carried out and are listed in The dependence of the mobile amorphous fraction on the degree of crystallinity of the semi-crystalline C-10-1 is shown in The change in the heat capacity for the fully amorphous material and the heat of fusion for the fully crystalline material were also obtained using the same method as for C-10-1. In ®30B are shown in −1. Moreover, there were bands of asymmetrical and symmetrical C-O bonds of the ester at 1246 and 1129 cm−1. Two bands of asymmetrical vibrations and symmetrical C-H bonds of the methyl and methylene groups were observed at 2990 and 2940 cm−1. However, no band was observed above 3000 cm−1. The FT IR spectra of the P3HB, PU400, and P3HB blends with 10 wt.%. of PU400 and its hybrid nanobiocomposites with Cloisite−1, which almost disappeared in the spectrum of the C-10-0. A wide band of valence vibrations of asymmetric and symmetric methylene groups of PU400 was observed in the range of 2800–3000 cm−1. The band of valence vibrations of the urethane carbonyl groups appeared at 1702 cm−1. Bands of the asymmetric and symmetric C-O bonds of the urethane groups are visible at 1271 and 1095 cm−1.In the FTIR spectrum of PU400 , there w−1, the intensity of which slightly increased with increasing nanoclay contents. In the 2800–3000 cm−1 range, there were three bands of asymmetrical and symmetrical C-H bonds of the methyl and methylene groups at 2877, 2933, and 2977 cm−1. A combined band of valence vibrations of the C=O ester and urethane groups was observed at 1707 cm−1. The common band for esters and urethanes for the vibrations of asymmetric C-O bonds appeared at a wavenumber of 1270 cm−1, and the band of symmetric vibrations of C-O bonds appeared at 1097 cm−1.In the FTIR spectrum of the hybrid nanobiocomposites , a small®30B nanoclay and linear aliphatic polyurethane were prepared with the aim of improving the physical properties of the P3HB matrix. Polyurethane (PU400) was obtained by reacting polyethylene glycol with a molecular weight of 400 g/mole with hexamethylene 1,6-diisocyanate (HDI). The introduction of two additives aimed to improve the biopolymer properties resulting from the plasticizing effect of polyurethane, as described earlier [Nanobiocomposites of P3HB modified with Cloisite earlier , and the®30B) led to a fully exfoliated structure (C-10-1). On the other hand, the presence of a larger amount of Cloisite®30B resulted in possible partial intercalation in addition to delamination .The addition of quantities greater than 1 wt.%, i.e., 2 wt.% or 3 wt.% of Cloisite®30B and 10 wt.% of PU400, whose impact resistance enhancement was due to the exfoliated structure and the formation of the P3HB-PU-MMT adducts as observed on the SEM micrographs. The addition of organomodified nanoclay resulted in a stiffening of the exfoliated structure and an increase in the hardness of the composite but below the hardness value of the unfilled P3HB.The obtained P3HB nanobiocomposites showed a predominance of an exfoliated structure, but a decrease in the content of nanofiller resulted in only the exfoliated structure, which had an impact on the mechanical properties. Therefore, the best mechanical properties were shown by the composite designated as C-10-1, containing 1 wt.% of Cloisite®30B compared to the unfilled P3HB. Increasing the amount of nanofiller not only failed to further increase the thermostability but even induced its decrease. Nanocomposites with Cloisite®30B alone were obtained by Naguib using an intercalation method. It was observed that the greater the addition of nanoclay, the greater the reduction in the composite decomposition temperature. It should be noted that 5 and 10 wt.% of Cloisite®30B were used, and only a deterioration in the thermal properties of the composites was observed compared to the neat P3HB [The thermal properties of the produced nanobiocomposites were improved as expected. The addition of the PU400 modifier caused an increase in the onset of the degradation temperature by 10 °C, and a further increase of 20 °C or 30 °C due to the addition of 1 wt.% of Cloisiteeat P3HB .®30B led to a decrease in the degree of crystallinity of nanobiocomposites in reference to P3HB. The X-ray analysis of the prepared nanobiocomposites also confirmed an increase in the amorphousness of the nanobiocomposites compared to the semi-crystalline unfilled P3HB.The addition of polyurethane caused the plasticization of P3HB, which appeared in the form of a decrease in the glass transition temperature, while the addition of nanoclay caused a stiffening effect and a further increase in the glass transition temperature as the nanoclay content increased. In summary, the plasticizing effect was not completely suppressed by the presence of nanofiller because the value of the glass transition temperature of the nanocomposites remained lower than the glass transition temperature of the unfilled P3HB. The tendency of the Tg to decrease has been observed in other studies ,42,43. FFrom the comparison of the melting point values of the individual nanobiocomposites and their degradation onset temperatures , it was This increases the possibilities of using P3HB for the production of disposable packaging materials or in medical applications, e.g., as implants, drug carriers, etc. Preliminary studies of nanofiber formation and active substance release involving P3HB and cytisine have already been carried out .®30B nanoclay in the P3HB matrix with the addition of aliphatic linear polyurethane (PU400) on selected thermal and mechanical properties of the produced nanobiocomposites.The conducted tests clearly showed the influence of the dispersion degree of Cloisite®30B, and delamination occurred with partial intercalation as the proportion of nanoclay in the matrix increased. This was possible due to the strong interaction between the components and the shear forces during direct mixing in a co-rotating twin-screw microextruder.The polymer matrix chains penetrated the interlayer spaces of Cloisite®30B. The polyester matrix modified with 10% PU400 and the smallest amount of Cloisite®30B (1 wt.%) was characterized by the formation of P3HB-PU-MMT adducts easily displaced with respect to each other, which positively affected the mechanical properties of the nanobiocomposites, i.e., better impact strength and lower hardness. The best mechanical properties were shown by a nanocomposite containing 1 wt.%. Cloisite®30B .The morphology studies on the produced nanobiocomposites showed the compatibility of the components used and the homogeneity of the obtained structure, the plasticizing effect of PU400, and the stiffening effect of CloisiteThe introduction of nanoplatelets into the polymer matrix resulted in better thermal stability of the nanocomposites as measured by the TGA method under non-oxidizing conditions. There was an increase in the degradation temperature by 30 °C. As the concentration of the nanoplatelets increased, the thermal stability of the nanocomposites deteriorated but remained better compared to the unfilled P3HB.Qualitative thermal analysis showed a decrease in the glass transition and melting temperature of the P3HB nanobiocomposites. A reduction of the melting temperature was beneficial due to easier processing and prevented material degradation.The obtained nanobiocomposites containing the smallest amount of nanofiller (1 wt.%) exhibited an exfoliated structure and formed P3HB-PU-MMT adducts, which were characterized by the best mechanical and thermal properties.


Heart failure with preserved ejection fraction (HFpEF) is increasingly recognized as a heterogeneous, systemic clinical syndrome. The complex nature of HFpEF makes it a diagnostic and therapeutic challenge. Recently, sodium-glucose cotransporter-2 (SGLT2) inhibitors have emerged as a promising therapy for HFpEF, as the EMPEROR-Preserved trial demonstrated that empagliflozin reduces the risk of the composite cardiovascular (CV) endpoint of death or heart failure (HF) hospitalization in HF patients with LVEF > 40%. Therefoet al provided valuable insights into these issues[HFpEF is considered a geriatric syndrome, as most patients with HFpEF are ≥ 65 years old with multiple comorbidities and limited functional status. Therefopost hoc analysis of the EMPEROR-Preserved trial, these investigators examined the treatment effect size of empagliflozin across pre-specified age subgroups. Participants were divided into four groups . Eighty percent (4789 out of 5988) of participants were ≥ 65 years old, representing a typical HFpEF patient population. Among them, 27% (1299 out of 4789) were ≥ 80 years old, a group rarely represented in previous trials. As expected, patients of older age had a greater burden of comorbidities, including higher blood pressure, lower eGFR, and higher prevalence of atrial arrhythmias. Advanced age was associated with increased incidence of the primary composite outcome of CV death or HF hospitalization. The investigators discovered that the relative risk reduction with empagliflozin was similar across the age groups , suggesting that the beneficial effect of empagliflozin is maintained across all age groups. Of note, prior analysis has suggested that empagliflozin has attenuated beneficial effects for patients with LVEF > 65%[In a Preservation of function and independence is one of the core values of geriatric care and an important goal for HFpEF management. EmpagliPolypharmacy is common in older patients, and the elderly are particularly susceptible to medication-associated side effects. The majority of participants in the EMPEROR-Preserved trial were concurrently taking commonly prescribed CV medications, including ARB/ACEi, beta-blockers, and statins, representing a typical population seen in clinical practice. As SGLTet al. provides evidence that empagliflozin is efficacious and safe in patients with HFpEF across all adult age groups, including the very elderly (≥ 80 years old)[etc.[In summary, the study by Bohm ears old). The benears old)–7,9 suggold)[etc.. Additio


Background: Heart failure with preserved ejection fraction (HFpEF) is prevalent in women and is associated with atrial fibrillation (AF). However, sex associations in AF-related HFpEF are not well explored. Aim: We studied differences between men and women with and without AF-related HFpEF symptoms on left ventricular (LV) geometry and diastolic dysfunction (DD) and their effect on cardiovascular events. Methods: Retrospectively, HFpEF patients with and without a history of AF referred for echocardiography were studied. Echocardiographic assessments were focused on LV geometry and diastolic functions. Patients were followed for the occurrence of cardiac events defined as death and cardiac hospitalization. Results: We studied 556 patients . Compared to HFpEF without AF (HFpEF-AF), HFpEF with AF patients (HFpEF+AF) were older , had more risk factors, comorbidities, left ventricular hypertrophy , higher relative wall thickness , and DD . HFpEF+AF women had the worst clinical, LV geometric, and diastolic functional profiles and highest rates of cardiovascular outcomes compared to HFpEF+AF men and were the only group to predict outcomes , while HFpEF-AF women were a low-risk group; HFpEF+AF and HFpEF-AF men had intermediate cardiovascular outcomes which were confirmed after propensity score matching. Conclusions: Among patients with HFpEF, women with AF had more abnormal LV geometry and diastolic function and had an increased risk of adverse cardiovascular outcomes independent of traditional risk factors, comorbidities, and baseline diastolic function. Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome that is associated with at least comparable morbidity and mortality to patients with heart failure with reduced ejection fraction (HFrEF). Despite the overlapping prognosis and clinical presentation, most of the medications used in HFrEF are not associated with clinical benefit in HFpEF, and the only medications that are associated with any level of clinical benefit do so modestly or only for symptom management. Risk factors and clinical phenotypes of HFpEF are significantly heterogeneous; however, atrial fibrillation (AF) has been linked to a specific high likelihood of development of left atrial enlargement, the elevation of left ventricular (LV) filling pressure (LVFP), diastolic dysfunction, subsequent development of HFpEF, and increased association with adverse cardiac outcomes. Epidemiologically, HFpEF is more prevalent in women than men, with nearly a 2:1 ratio. In randomized clinical trials concerned with heart failure, men represent the majority of the studies concerned with HFrEF, while the representation in randomized clinical trials in patients with HFpEF seems to be mostly by women. The PARAGON-HF study increased the interest in the sex-specific differences in HFpEF when it was found that the only group of HFpEF patients that showed clinically meaningful response to sacubitril/valsartan therapy was women; however, the reasons behind that are not yet explored /BSA, where LVEDD is left ventricular end-diastolic dimension, IVSd is the interventricular septal dimension, and PWd is posterior wall dimension. All geometrical measurements were done at end-diastole from the parasternal long-axis window 1 mm below the mitral valve tipsRWT = 2 × PWd/LVEDD, where PWd is the posterior wall dimension, and LVEDD is the left ventricular end-diastolic dimension. LVMi was calculated using the formula LVMi = [0.8 × (1.04 × (((LVEDD + IVSd +PWd)The pulsed-wave Doppler-derived transmittal velocity and spectral tissue Doppler-derived mitral annular velocity were obtained from the apical 4-chamber view. The early diastolic wave velocity (E) and the late diastolic atrial contraction wave velocity (A) were measured using pulsed-wave Doppler recording, and the early diastolic mitral annular velocity (e’) was measured from the septal mitral annular positions. The E/e’ ratio was calculated to assess LV filling pressure (LVFP) for all patients. In addition, the peak tricuspid valve regurgitation velocity was measured, and the presence of diastolic dysfunction was assessed as recommended by the American Society of Echocardiography guidelines. All measurements were made in ≥3 consecutive cardiac cycles, and average values were used for the final analyses.Patients were followed for a median of 1.1 years (13 months) for cardiovascular death or hospitalization for cardiovascular causes. Events were recorded by chart review and by means of telephone call contact with patients. Patients were checked for the prediction of outcomes based on their sex, presence of a history of AF or elevated LVFP at the time of the study. t-test or analysis of variance (ANOVA) if they are normally distributed or the Mann–Whitney U test if they are not normally distributed. Cox regression and Kaplan–Meir survival curves were used to assess the predictability of outcomes in different subgroups. Categorical data are presented as numbers (%) and were compared using the chi-square test. Continuous data are presented as mean ± SD. Data were tested for normality using Kolmogorov-Smirnov and Shapiro-Wilk tests and, accordingly, continuous data were compared using a A 1:1 propensity score matching was conducted to match patients with AF to patients without AF. The propensity score was done using binominal logistic regression with the independent variables (covariates) being age, risk factors , presence of co-morbidities , and absolute parameters of diastolic functions . A 1:1 propensity score matching was done using the nearest neighbor classification and resulted in 156 matched patients (78 in each group)p < 0.05. All analyses will be performed with commercially available software .Differences were considered statistically significant at During the specified period, 556 patients were retrospectively studied. Compared to HFpEF men, HFpEF women showed no statistically significant difference in age, or cardiac risk factors, including a history of diabetes mellitus, hypertension, and hyperlipidemia, except for more smoking in men. Regarding comorbidities, more men had chronic obstructive pulmonary disease (COPD) and worse renal functions, as suggested by serum creatinine, while women were slightly more anemic, as suggested by hemoglobin levels.Compared to HFpEF patients without AF (HFpEF-AF), HFpEF patients with AF (HFpEF+AF) were older and had more cardiac risk factors except for similar history of diabetes. Regarding comorbidities, HFpEF+AF patients had worse renal function and were more anemic but had a similar history of COPD compared to HFpEF-AF patients.The overall and subgroup description of echocardiographic comparisons of structural and functional characteristics are summarized in Compared to HFpEF men, HFpEF women were found to have higher EF values, lower LV end-diastolic volume index (LVEDVi), lower LV end-systolic volume index (LVESVi), similar LAVi, and relative wall thickness (RWT), lower left ventricular mass indexed to body surface area (LVMi) but similar prevalent LV hypertrophy. HFpEF women were found to have similar absolute diastolic variables compared to HFpEF men, namely E-wave velocity, e’ velocity, E/e’ ratio, and TRV velocity, with the exception of higher A wave velocity translating into lower E/A ratio, and a shorter E-wave deceleration time. The occurrence of diastolic dysfunction was also not different between men and women.On the other hand, compared to HFpEF-AF patients, patients with AF were found to have similar EF, LVEDVi and LVESVi; however, they had higher LAVi, RWT, and LVMi. Diastolic parameters were statistically significantly worse in HFpEF+AF compared to HFpEF-AF, namely higher E-wave velocity, higher E/A ratio, lower e’ velocity, higher E/e’ ratio, and higher TRV velocity, except for similar A wave velocity and E-wave deceleration time. Diastolic dysfunction was diagnosed significantly more in HFpEF+AF.The demographic, clinical, and echocardiographic comparisons of HFpEF women and men with and without AF are summarized in Briefly, age, cardiac risk factors, comorbidities , EF, LAVi, and echocardiographic measures of geometry and diastolic function were significantly different between sexes with and without AF. HFpEF-AF women represented the lowest risk profile among all these parameters. Furthermore, HFpEF+AF women had the worst demographic and clinical profiles in terms of being older, having the most history of hypertension and hyperlipidemia, having the highest prevalence of COPD, having worse renal function, and having lower hemoglobin levels. In terms of echocardiographic parameters, HFpEF+AF women were found to have the smallest LVEDVi and LVESVi, the largest LAVi, the worst RWT and LVMi, the lowest septal e’ velocity and E/e’ ratio, the highest TRV velocity, and higher prevalent diastolic dysfunction than HFpEF+AF men. HFpEF+AF men had a higher history of diabetes and smoking, worse renal function, higher E-wave velocity and E/A ratio and shorter E-deceleration time while sharing a similar high-risk profile with HFpEF+AF women with regard to a history of COPD, hemoglobin levels, and E/e’ ratio.p = 0.291; p = 0.012, 0.008).In the study follow-up period, 25 patients died, 91 patients were hospitalized for cardiac causes, and 99 patients had the composite of death or cardiac hospitalization . There wp = 0.036, 0.018 and 0.014, all p < 0.05, respectively; HFpEF+AF women had the highest rates of death, cardiac hospitalization, and composite outcomes  were women, and 16% of the overall group had a history of AF. Second, while HFpEF women did not differ compared to men in the overall clinical or demographic parameters and did differ in adverse cardiac events, AF was more common in HFpEF men and was associated with worse demographic, clinical and echocardiographic measures of geometry and diastolic function and worse adverse cardiovascular events. Finally, HFpEF+AF women had worse clinical, LV geometric and diastolic functional profiles and were associated with a higher risk of adverse events, while HFpEF-AF women were a low-risk group, and HFpEF+AF and HFpEF-AF men were the intermediate groups.Epidemiological studies suggest that women predominate in the incidence of HFpEF with a women-to-men ratio that approximates 2:1 . This woHFpEF is known to be associated with changes in ventricular geometrical properties and cardiac remodeling that become more pronounced with aging, in hypertensives, as well as with AF . The resAccording to recent studies, there are no overall differences in clinical outcomes between HFpEF women and men. In our study as well, the overall risk of outcomes was not different between men and women. It is to be highlighted that, in some studies, women with HFpEF carry a higher risk of outcomes after adjustment of covariates , suggestEpidemiologically, there is a strong relationship between HFpEF and AF. The relationship seems to be bidirectional, where patients with AF have an elevated risk of developing HFpEF and, conversely, most patients with HFpEF eventually develop AF . Such HFIn our study, we confirmed such a high-risk association when we found that the presence of a history of AF is a very strong predictor of clinical, geometrical, and hemodynamic alteration in HFpEF patients, as well as a predictor of LV diastolic functions. Moreover, AF was a significant predictor of adverse cardiac events.p = 0.002), a finding that is in agreement with prior reports that confirm a 1.5 to 2 increased risk of AF in men compared to women [In our study AF was more prevalent in men compared to women (relative risk 1.81, 95% CI: 1.23–2.64, A number of observational studies have demonstrated the association between female sex and the risk of AF-related stroke and thromboembolism . HoweverWhile traditional risk factors still affect myocardial function similarly in women and men, women exhibit sex-specific physiological and pathological elements beyond traditional risk factors that may independently cause differences in progression, clinical presentation, and outcomes of myocardial dysfunction, as well as the development of AF. Some of these women-specific risk factors include the lack of protective estrogen in the perimenopausal period or estrogen receptor dysfunction, pregnancy, breast cancer-related treatments, specific responses to psychological stressors and a greater prevalence of autoimmune diseases .Such sex-specific elements can be linked to differences in the effects of therapeutics between women and men, such as that observed in the PARAGON-HF trial . MoreoveDespite the fact that women a smaller left atria and ventricles compared to men, as shown in our study, small magnetic resonance studies have shown that atrial fibrosis in patients with HFpEF was detected in women more than in men  and thatIt’s important here to note that, despite that such effects are recognized in the literature, they are mostly driven by retrospective and post hoc sex-based analyses. The translation into clinical practice is limited by the underrepresentation of women in clinical trials and the lack of sex-specific prospective studies.This is a retrospective study with a relatively small number of patients, and larger prospective studies will need to be done to confirm the study findings. The plasma levels of brain natriuretic peptide were not measured in this study as patients were assessed on an outpatient basis and should be measured to detect differences in future studies. In our study, there was no assessment of the left atrial anatomic substrates and fibrosis and no assessment of LA or LV mechanical behavior, such as strain and strain rate measurements which could further elucidate differences between men and women. Moreover, heart rate is a known parameter to affect echocardiographic measurements in patients with atrial fibrillation, but heart rate data was not available in our study. However, in our laboratory, and as recommended by the American society of echocardiography, measurements in patients with atrial fibrillation are averaged from at least three cardiac cycles. Finally, despite the rigorous matching done for clinical and echocardiographic covariates, there is a possibility the variables unaccounted for that affect clinical outcomes and diastolic properties may influence the predictive ability of our models. As such, the models used in our study were done for qualitative exploratory purposes and model validation was not attempted. Therefore, future prospective analyses should be performed to confirm our findings and validate the models used.AF carries a special risk for the development of outcomes in patients with HFpEF. Despite the fact that AF is more common in men, women with AF are a special group of patients with worse clinical, left ventricular geometrical, and diastolic profiles and were the only group in our study to be associated with adverse clinical outcomes. As such, women with AF-associated HFpEF are at a particular increase of adverse cardiovascular outcomes independent of traditional risk factors, comorbidities and baseline diastolic function, a finding that seems more pronounced when compared to men. Further studies are needed to determine the underlying pathophysiology and to identify novel preventive and disease-modifying treatments for HFpEF+AF.


Heart failure (HF) with preserved ejection fraction (HFpEF) prevalence is increasing, and large clinical trials have failed to reduce mortality. A major reason for this outcome is the failure to translate results from basic research to the clinics. Evaluation of HFpEF in mouse models requires assessing three major key features defining this complex syndrome: the presence of a preserved left ventricular ejection fraction (LVEF), diastolic dysfunction, and the development of HF. In addition, HFpEF is associated with multiple comorbidities such as systemic arterial hypertension, chronic obstructive pulmonary disease, sleep apnea, diabetes, and obesity; thus, non-cardiac disorders assessment is crucial for a complete phenotype characterization. Non-invasive procedures present unquestionable advantages to maintain animal welfare and enable longitudinal analyses. However, unequivocally determining the presence of HFpEF using these methods remains challenging.Transthoracic echocardiography (TTE) represents an invaluable tool in HFpEF diagnosis, allowing evaluation of LVEF, diastolic dysfunction, and lung congestion in mice. Since conventional parameters used to evaluate an abnormal diastole like E/A ratio, isovolumic relaxation time, and E/e′ may pose limitations in mice, including advanced TTE techniques to characterize cardiac motion, including an assessment under stress, will improve diagnosis. Patients with HFpEF also show electrical cardiac remodelling and therefore electrocardiography may add valuable information in mouse models to assess chronotropic incompetence and sinoatrial node dysfunction, which are major contributors to exercise intolerance. To complete the non-invasive diagnosis of HF, low aerobic exercise capacity and fatigue using exercise tests, impaired oxygen exchange using metabolic cages, and determination of blood biomarkers can be determined. Finally, since HFpEF patients commonly present non-cardiac pathological conditions, acquisition of systemic and pulmonary arterial pressures, blood glucose levels, and performing glucose tolerance and insulin resistance tests are required for a complete phenotyping.Identification of reliable models of HFpEF in mice by using proper diagnosis tools is necessary to translate basic research results to the clinics. Determining the presence of several HFpEF indicators and a higher number of abnormal parameters will lead to more reliable evidence of HFpEF. About han HFpEF , 15. As Non-invasive HFpEF diagnosis remains challenging in humans , even afMice are the most widely used laboratory animal in translational research and they are also the most popular animal when trying to model human HFpEF . ConsideNon-invasive assessment in mice enables repeated analysis of multiple parameters while causing minimal discomfort to animals. Apart from the evident agreement with the 3Rs posing a more ethical and responsible care and used of animals, it also enables to better demonstration of similarities with the human phenotype, as in persons hardly ever invasive procedures are performed to diagnose cardiac function and HF. For this reason, this work aims to address the key steps in non-invasive diagnosis of HFpEF in mouse models, with a main focus on non-invasive imaging techniques, as they represent a gold standard in the evaluation of HFpEF in human patients. We also highlight gaps and future directions for a global assessment of these mouse models.Transthoracic echocardiography (TTE) and cardiac magnetic resonance (CMR) are the non-invasive imaging techniques most commonly employed for the assessment of HFpEF , 21. EchIt is important to remark that cardiac function and size are highly dependent on the mouse strain . ConsideLVEF is the fraction of blood ejected from the left ventricle (LV) during one heartbeat, expressed as a percentage . LVEF is3)/(2.4 + EDD) and ESV = (7 × ESD3)/(2.4 + ESD) [2)/(3 π ventricular length) and ESV = (0.85 × (ESA2)/(3π ventricular length )[There are several methods to obtain LV EDV and ESV using TTE. The simplest and fastest method is to calculate the volumes according to Teichholz formula (single plane), acquiring images in a short axis view (SAX) in M-mode Fig. A and dis4 + ESD) , 29. The4 + ESD) . For a m length ). Another length ). The LV  length ). More so length ). Neverth length ), 32.Fig.CMR provides high accuracy and reproducibility in the calculation of ventricular volumes . To calcDiastolic dysfunction is the fundamental feature of HFpEF, characterized by abnormal LV filling due to an impaired LV relaxation and increased LV stiffness, leading to high LV pressures . In contThe mitral inflow pattern in mice can be assessed by using an apical 4-chamber view applying pulse wave Doppler echocardiography (PWD) and placing the sample volume in the LV, close to the valve  in the apical 4-chamber view in mice by placing the sample volume at the septal corner of the mitral annulus. Two main waves are generated according to myocardial diastolic movement: E′ wave, during early and passive filling and A′ wave, during the active filling. The peak velocity of each wave is then determined  changes in HFrEF have been widely studied in human patients; however, studies in HFpEF patients are scarce , 53. As Stress ECG represents a valuable tool to highlight pathological features associated with exercise intolerance. In human patients, a poor response of the heart rate to exercise (chronotropic incompetence) and a functional limitation to further sinus node activation during exercise  would explain at least in part this impaired exercise capability , 58. TheHFpEF diagnosis can be challenging in humans due to non-specific symptoms like fatigue , and thiNon-invasive approaches for HF identification in mice usually encompass some of the following measurements: low aerobic exercise capacity, impaired oxygen exchange, and lung congestion , 15, 60.Exercise intolerance is a hallmark of HF , and its2max) represents the greatest possible amount of oxygen supplied during exercise [Gas exchange data is compromised by subtle lung congestion during HF leading to exercise intolerance, even if findings are normal at resting conditions. Peak oxygen consumption  and amino-terminal pro-brain natriuretic peptide (NT-proBNP) are both increased in the presence of elevated LV filling pressures, and they are the most widely used biomarkers to assess HF. However, these markers are most useful in acute HF and their clinical value in chronic HF is less clear . In thisCommon abnormal conditions shown in HFpEF patients include systemic arterial hypertension (SAH), chronic obstructive pulmonary disease (COPD), sleep apnea, diabetes, and obesity. Ideally, the diagnostic tools used in murine models should be similar to those used in human medicine to get a more accurately translational approach.In systemic arterial pressure evaluation, since systemic arterial hypertension (SAH) alters the heart’s function and structure, elucidating mechanisms involved in the development of HFpEF necessarily requires monitoring blood arterial pressure. In conscious rodents, the most widely used indirect method for monitoring this parameter is the cuff technique placed in the tail, which determines the cuff pressure at which changes in blood flow occur during occlusion or release of the cuff . ResearcIn noninvasive assessment of pulmonary arterial hypertension (PAH), echocardiography of the pulmonary artery (PA) dynamics is the main tool for non-invasive assessment of PAH in mice , 88. TheIn the assessment of hyperglycaemia, glucose tolerance, and insulin resistance, accurately performing metabolic tests relies on proper mouse preparation selected careful selection of the protocol. Different fasting and drug administration protocols have been described and researchers must consider pros and cons before choosing the most appropriate one for their model .Measuring glucose levels in mice is simple and fast using a glucometer. The coccygeal vein can be used to obtain a drop of blood after a fasted period . High faIn multiorgan assessment, another important consideration is the analysis of different organs in the development of HFpEF, which is seldom reported. It is widely recognized that human HFpEF, rather than being characterized by an isolated abnormality in LV diastolic function, is a heterogeneous systemic syndrome characterized by multiple cardiac, vascular, and peripheral pathological features. Accordingly, patients show cumulative risk factors and comorbidities , 39, 92.Using proper diagnostic tools in animal models is essential to improve the translation of basic research results to clinical medicine. Highlighting the three key points in HFpEF, preserved ejection fraction, diastolic dysfunction and HF is crucial to evidence a murine model of this syndrome. Since no single parameter can establish the presence of diastolic dysfunction and HF unequivocally, researchers must support the diagnosis by assessing several parameters simultaneously. Finally, to better recapitulate the complexity of this syndrome, a broad evaluation including typical comorbidity conditions associated to HFpEF in humans is crucial.


Cholesteatoma may cause bone erosion, with high morbidity and mortality rates. Tumor Necrosis Factor - Alpha (TNF-a) is one of the main cytokines involved in this process. Our goal was to evaluate the role of TNF-a in Bone Resorption and its effect on cholesteatoma.analysis and critical literature review.Different studies have demonstrated that TNF-a is capable of causing bone erosion. It may stimulate the differentiation and maturation of osteoclasts or it may act on the bone matrix, exposing it to the action of the osteoclasts. It is possible to inhibit TNF-a, reducing its effects and prevent bone loss in illnesses such as rheumatoid arthritis, and there has been no specific investigation regarding cholesteatomas. All studies agree on the importance of TNF-a in the bone resorption process present in cholesteatomas, and on the degree of destruction observed; however, there is no consensus as to its location. These differences are probably due to receptor site.TNF-a, present in cholesteatomas, promotes bone resorption, along with other cytokines (RANKL and IL-1) related to complications. Acquired middle ear cholesteatoma was first described by Curveilhier in 1829. It is characterized by the invasion of keratinized stratified squamous epithelium into the tympanic cavity. This epithelium is different from that usually found in the middle ear.Cholesteatoma may cause bone destruction and result in intratemporal and intracranial complications,There are numerous theories to explain the destructive properties (erosion) of cholesteatomas. Pressure due to accumulated keratin and other waste was initially proposed as the cause of destruction. A biochemical theory eventually was postulated, in which enzymes and cytokines released by cholesteatomas would cause bone lysis and destruction.Cytokines are a group of low molecular weight proteins for communication between cells.The aim of this paper is to analyze the importance of tumor necrosis factor alpha (TNF-α) in bone resorption and its action in acquired middle ear cholesteatoma based on a review and an analysis of literature.TNF-α is produced by activated macrophages, lymphocytes or monocytes.When released at low concentrations, TNF-α acts on endothelial cells to promote vasodilation and stimulates these cells to secrete a group of leukocyte-chemotaxis cytokines named chemokines. Resulting local inflammation combats infections.TNF-α leads to bone destruction by acting directly on osteoclast differentiation and maturation, and by indirectly exposing the bone matrix.TNF-α may be inhibited by antagonist solutions containing a soluble portion that block its receptors. Assuma et al.The first papers that investigated the role of TNF-α in cholesteatoma were published in the beginning of the 1990s.Yan and HuangMarenda and AufdemorteAmar et al.Chung and YoonSastry et al.Akimoto et al.Yong-Soo et al.Yetiser et al.Alves and RibeiroCytokines are proteins formed within cells that, when released, interact with transmembrane cell receptors to produce their biological effect.TNF-α is produced by activated macrophages or by lymphocytes and monocytes. After linking with its receptors, TNF-α stimulates transcription and production of the IκB kinase enzyme, which in turn produces the nuclear factor κB (NF-κB). When activated, NF-κb acts on the cell nucleus inducing the production of a variety of proteins involved in inflammation and immunological processes responsible for the biological action of TNF-α.Studies on the effect of TNF-α in cholesteatoma started in the beginning of the 1990s, when TNF-α was observed in that condition.16,17 Yan and HuangOsteoclasts are the main cells involved in bone resorption. They originate from monocytes and macrophages and act directly on the bone matrix, causing bone erosion and remodeling. TNF-α, IL-6, IL-1 α or β, interleukin 11 (IL-11), and the macrophage colony stimulating factor (M-CSF) are all responsible for osteoclast differentiation and regulation. These factors, therefore, act directly on bone resorption.Other factors, however, have an indirect action on osteoclasts or on bone-lining cells, by exposing the bone matrix to osteoclasts. These include the parathyroid hormone, prostaglandins, estrogen, IL-11, IL-1 α or β, and TNF- α (which acts both directly and indirectly).Osteoblasts release a substance - RANKL - that stimulates differentiation of macrophages into osteoclasts. There is synergy between TNF-α and RANKL in that both cooperate to increase the action of osteoclasts.11 Authors that have studied TNF-α action mechanisms believe that it promotes bone resorption by two pathways (direct and indirect),All of the authors we reviewed agree that TNF-α is present in cholesteatomas. There is no consensus, however, about its localization. Alves and RibeiroThe quantity of TNF-α, and not only the number of receptors, also seems to be related to bone destruction. Various authors have attempted to relate TNF-α levels with intraoperative findings.Based on these papers, we may conclude that TNF-α is associated with bone destruction and defines its severity.TNF-α is one of the main factors responsible for bone resorption and resulting complications in a variety of diseases. The possibility of interrupting the inflammation seen in such chronic diseases was raised following the discovery of natural inhibitors against TNF-α, which could reduce morbidity and mortality.In periodontitis injection of a solution containing a TNF-α antagonist (s NF RI) inhibits osteoclast formation, reduces inflammatory cell recruitment and reduces bone loss by 60%.Anti-TNF-α antibodies can also reduce and postpone bone destruction in rheumatoid arthritis.TNF-α is found in cholesteatomas, promoting bone resorption by different routes. It acts on osteoclast differentiation and maturation (direct pathway) and also exposes the bone matrix (indirect pathway). TNF-α, however, is not the only factor involved in bone resorption; it acts in association with interleukin 1 and RANKL. Furthermore, local levels of TNF-α are related with the degree of bone destruction in this disease.