The purpose of this study is to evaluate the wear and fracture strength of crowns and three-unit partial fixed dental prosthesis (FDP) fabricated using by Bruxzir and Incoris TZI as recently introduced monolithic zirconia materials.n = 8). All specimens were subjected to a 2-body wear test in a dual axis chewing simulator for 1,200,000 loading cycles against steatite antagonist balls. The fracture strength and volumetric loss were recorded. The obtained data were statistically analyzed by 2-way ANOVA testing (α = 0.05).A total of sixteen crowns and sixteen three-unit FDPs were fabricated using Bruxzir and Incoris TZI (p < 0.05). Of the two monolithic systems, Incoris TZI exhibited more wear than Bruxzir. The fracture strengths of Bruxzir crowns and FDPs were found to be higher than those of the crowns and FDPs fabricated with Incoris TZI (p < 0.05).The mean volumetric loss of the crowns was higher than that of the three-unit FDPs (In in vitro test conditions, Bruxzir and Incoris TZI monolithic zirconia systems are fracture-resistant for the crown and FDP application against physiologic chewing forces owing to dynamic aging. Among newly developed monolithic zirconia materials, Bruxzir is found to be more resistant to fracture compared to the Incoris TZI. Esthetic expectations are the main reason for preferring ceramic restorations, for which the usual processing method is veneering. Major problems associated with multilayered restorations are their low fracture strength and surface chipping. Therefore, new processing techniques have been developed to resolve the chipping problem encountered with ceramic veneering layers . For exaMonolithic restorations aim at improving the final quality of restorations. Further, the problems of surface flaws and chipping problems encountered with veneering can be resolved using monolithic zirconia restorations . ZirconiPhysiologic chewing forces are in the range of 10–120 N, while parafunctional forces are greater in the range of 200–800 N –8 which Monolithic zirconia restorations are not preferred when the esthetic function is the priority. However, these systems are beneficial in the case of fixed dental prostheses supported by pathological attrition or severely damaged teeth (FDPs). Adhesive bonding of monolithic restorations is beneficial in various clinical situations, such as excessive unloading forces, compromised mechanical retention, and limited space for adequate tooth preparation . FurtherThe high fracture strength of yttria-stabilized zirconia (YSZ) is attributed to the physical properties of partially stabilized zirconia. In previous studies, the fracture strength of YSZ was reported to vary from 900 N  to 2000 Preclinical evaluations help to determine the physical and mechanical behavior of materials. Although the fatigue testing standards (DIN EN ISO 22674) of fixed dental prosthesis materials are established under certain test conditions, it is controversial how much of the intraoral conditions are accurately represented by these standards , 28. ResThe aim of this in vitro study is to evaluate the wear and fracture strength of crowns and three-unit partial FDPs fabricated using recently introduced monolithic zirconia materials and subjecting them to 1,200,000 chewing cycle versus steatite balls. The null hypothesis tested was that no difference would be detected in the wear and fracture strength properties of different tested materials.n = 8). Bruxzir crowns and three-unit FDPs were fabricated using monolithic zirconium blanks  designed using a Cerec inLab MC X5 system . Incoris TZI crowns and three-unit FDPs were fabricated from monolithic blocks (40/19 = 40x19x15.5 mm) and designed using a Cerec inLab MC X5 system . The chemical composition, according to the manufacturer’s declaration of investigated Y-TZP ceramics is shown in Table 2 was selected for FDPs as recommended by the manufacturers. Bruxzir restorations were sintered at a temperature of 1580 °C for 2 h and then glazed with Bruxzir spray glaze powder  at a temperature of 830 °C according to the manufacturer’s instructions. Incoris TZI restorations were sintered at a temperature of 1510 °C for 2 h and then glazed with Cerec speed glaze spray  at a temperature of 750 °C according to the manufacturer’s instructions. All the restorations and preparations were carried out by the same dentist. Eight crowns and FDPs were created with the two different zirconia materials randomly.In the present study, a mandibular left first molar tooth of the dentulous mandibular cast  was selected for producing monolithic crown restorations. A mandibular left second premolar tooth  and a mandibular left second molar tooth  were selected for fabricating the FDPs. The selected teeth were prepared according to the accepted tooth preparation principles using a chamfer diamond rotary instrument  by adjusting for a 1 mm circumferential chamfer margin, 1.5 mm occlusal reduction, 1 mm axial preparation, and 6° convergence angle. After preparation, the master casts were evaluated using a surveyor to detect undercuts. The prepared teeth were then duplicated as master dies made of Ni-Cr by laser sintering. In total, thirty-two master model dies were obtained, including sixteen master casts that were made as crowns and sixteen master casts that were made as three-unit FDPs; the model dies were fabricated with Bruxzir  and Incoris TZI   were mixed and applied to the intaglio surfaces of the restorations according to the manufacturer’s instructions. The restorations were seated onto the dies and held in place by the application of finger pressure. Subsequently, the restorations were cured using a curing light for 20 s. Excess cement was removed with sponge pellets before curing and an air-blocking gel  was applied during the setting of the resin cement over 3 min. The obtained specimens were stored for 24 h at 37 °C before being subjected to dynamic aging.All the restorations were adhesively luted on Ni-Cr master cast dies using a dual cure composite material  according to the manufacturer’s instructions. The master cast dies were sun-blasted with 50 μm Al3) in all the specimens was calculated  from the marginal finish line of the restorations to 2-mm apical direction for the purpose of simulating the physiologic mobility of teeth. The metal dies were immersed in a wax bath, which was replaced by polyether in a second fabrication process, as previously described . Later, restorations on master cast dies were fixed in a resin mold, which acts as the sample holder for the chewing simulator, using a self-curing acrylic resin material . The specimens underwent thermocycling for 10,000 cycles between 5 and 55 °C over a dwell time of 60 s and a transfer time of 10 s . After thermocycling, the specimens were subjected to a 2-body wear test in a dual axis chewing simulator . Steatite ceramic balls  of 6 mm diameter were used as the antagonistic abraders. The balls were fixed to the upper sample holders of the chewing simulator using a light-curing composite resin . The chewing simulation parameters used are summarized in Table Following the aging procedure, the specimens were tested on a universal testing machine  until fracture. They were subjected to a compressive force at a crosshead speed of 1 mm/min with a round shaped modified bur of 4 mm diameter. A metal bar was positioned parallel to the long axes of the crown specimens and the buccal and lingual cusps of the crowns were used to apply the force. Force was transferred to the occlusal connector area of the FDP specimens. The maximum load necessary to fracture each specimen was recorded in Newtons (N).To characterize the surface wear patterns, selected specimens were evaluated by a scanning electron microscopy , for which the sample surfaces were initially coated with a thin layer of gold. The surfaces were then examined at a magnification of 100X at 25 keV.p < 0.05).Statistical analysis was performed using SPSS 20.0  for Windows. Having assessed that all the obtained results were normally distributed, the wear and fracture load data were analyzed by two-way ANOVA. Bonferroni adjustment was used for multiple comparisons. Two methods and two monolithic zirconias were used for 4 groups with 80% power, 5% margin of error and effect size of 0.65 with 8 samples in each group, totaling 32 samples. The sampling volume was obtained with the help of the program G * power 3.1.2. The results are expressed as a mean ± standard deviation and the level of significance is set at 5% (3) of the monolithic zirconia specimens is shown in Table p = 0.003). The mean volumetric loss of the crowns was observed to be higher than that of three-unit FDPs (p < 0.05). Of the two tested monolithic systems, Incoris TZI exhibited more wear than Bruxzir.The mean volumetric loss mm of the mp < 0.05). Moreover, Bruxzir FDPs exhibited significantly higher fracture strengths (4506.25 ± 166.44 N) than Incoris TZI FDPs (3327.13 ± 185.81 N) (p < 0.05). Besides, no statistically significant differences could be observed between the Bruxzir crowns and FDPs (p > 0.05). Representative SEM images of the Bruxzir and Incoris TZI crowns are shown in Fig. None of the samples fractured during dynamic aging. The mean fracture strength (N) of the monolithic zirconia is shown in Table This in vitro study evaluated the wear and fracture strength of crowns and FDPs fabricated using two recently introduced monolithic zirconia materials. The null hypothesis tested in the present study, which assumed no difference in terms of the wear and fracture strength properties between the two tested materials, was rejected.3), IPS e.max CAD (0.355 mm3), and Vita Mark II (0.472 mm3) were similar to that of human enamel (0.393 mm3). However, in the present study, 6 mm-thick disk-shaped specimens and a zirconia antagonist abrader were used. Moreover, the ceramic materials tested in this study exhibited lower hardness than Bruxzir and Incoris TZI monolithic systems.Zirconia has been developed with the aim of providing a stronger material for prosthetic dentistry. Ideal restorative materials should exhibit wear properties similar to those of human enamel and should not cause excess antagonist wear. Although short-term data is available on zirconia FDPs, a recent study showed that monolithic polished zirconia crowns caused less wear on antagonist enamel than glazed ceramic metal crowns . In a stParafunctional chewing forces are approximately ten times greater than physiologic chewing forces –8. Day bThe dynamic aging and fracture resistance of monolithic zirconia systems were determined by loading crowns and three-unit FDPs using an SD mechatronic chewing simulator . Heintze et al.  reportedIt has been reported that almost all materials that have any geometrical shape such as composite, natural teeth, metal, ceramic or steatite can be examined with LAS 20 laser scanner . AdvanceD’Archangelo et al.  reported2 connector size. No specimen fractured during dynamic loading, similar to the present study. The dimensions of the connector area are crucial for determining the strength of FDPs. In the current study, a connector size of 9 mm2 was selected according to the manufacturer’s suggestion. Apart from the connector design [On the basis of the obtained findings, almost all the tested monolithic zirconia materials exhibited high load strengths. In a previous study, the fracture strength of YSZ was reported to be in the range of 900–1200 N . In anotr design , the frar design , and user design . In the r design , 31, 34.The major limitation of this study is the difficulty of determining the ideal chewing cycle. In this regard, Özcan and Jonasch , in a syA second limitation of the present study is the lack of a secondary higher force against Bruxzir. Considering that Bruxzir material was originally produced against bruxism, bruxzir would have exhibited more strength than 49 N. However, the applied force in the present study was 49 N which is accepted as a normal chewing force in the posterior region, was used for Bruxzir and Incoris TZI restorations. Further studies may be carried out considering chewing forces specific for bruxers.Based on the findings of this in vitro study, both the monolithic zirconia crowns showed a small but significantly increased volumetric loss compared to three-unit FDPs. Of the two tested monolithic systems, Incoris TZI exhibited greater wear than Bruxzir. The fracture strengths of Bruxzir crowns and FDPs were found to be greater than those of their counterparts fabricated with Incoris TZI. Bruxzir and Incoris TZI monolithic zirconia systems were found to be fracture-resistant for crowns and FDPs against physiologic chewing forces owing to dynamic aging in vitro test conditions.


Early technical complications after pancreas transplantation are almost always unsalvageable. The two most common complications are vascular thrombosis and duodenal anastomotic leaks. We present a case of a duodenal stump leak that led to a large abscess and severe sepsis. The pancreas was salvaged by repairing the leak and creating a proximal diverting ileostomy. Several months later, the ileostomy was reversed. This was done by creating a defunctionalized Roux limb to exclude the pancreas. The patient healed well and continued to enjoy excellent glucose control. Whole pancreas transplantation has become an accepted form of treatment for type I diabetes mellitus. Based on Organ Procurement and Transplantation Network (OPTN) data as of May 22, 2019, 8,789 pancreas and 23,959 simultaneous kidney-pancreas transplants have been performed since January 1988. In 2018, there have been 192 pancreas and 836 kidney-pancreas transplants reported. Early graft loss is rare but is generally related to technical complications. Such complications are most commonly related to vascular thrombosis and intestinal anastomotic leaks. Traditionally, these complications are grave and graft salvage is not attempted. Retransplantation is also seldom attempted given the morbidity of the first operation.We present a single case experience where a duodenal stump leak occurred early after pancreas transplantation. Transplant pancreatectomy was successfully avoided using a nuanced technique.The patient is a middle-aged female with type I diabetes mellitus. She suffered from difficulties with glucose control and hypoglycemic unawareness. She also developed the sequelae of diabetes, including retinopathy and peripheral neuropathy. As a result, the patient received a pancreas transplant. The patient had preserved kidney function with a glomerular filtration rate (GFR) of 107 ml/min and a protein to creatinine ratio of 138 mg/g. Vascular reconstruction was performed using a standard donor iliac Y-graft to the superior mesenteric and splenic arteries on the graft end. The common iliac artery of the donor was reconstructed to that of the recipient. The donor portal vein was anastomosed into the inferior vena cava. The duodenum of the donor was then drained using a standard two-layer, hand-sewn anastomosis to the recipient ileum. Initially, the patient had an uneventful postoperative course. About a month later, she presented with increasing pain and diarrhea. Cross-sectional imaging revealed an abscess Figure . An explShe was found to have a large abscess cavity, and after adequate drainage, we did not find a leak at the duodenal anastomosis. The staple line at the proximal duodenum, however, had a 1-cm gap in it. The graft was initially thought to be unsalvageable. The edges of the leak were inflamed and devitalized. With intestinal contents passing by, any primary repair was guaranteed to fail. There was also not a precedent for a diversion this early after transplantation. We eventually decided to repair the defect with running non-absorbable monofilament stitches and performed a diverting loop ileostomy Figure . The patPancreas transplantation has long been the most effective therapy for type I diabetes mellitus -3. This The surgical complications of pancreas transplantation have been well-described -11. The The principle of enteric diversion for a leak is a well-established one. It has been long-utilized in perforated diverticulitis and perforated duodenal ulcers -15. The Duodenal leaks are devastating complications after pancreas transplantation. Drainage, repair, and diversion can be utilized to salvage the transplant. These principles are applicable to other types of intestinal leaks, such as perforated acute diverticulitis, as well. This case report illustrates that two-staged diversion followed by revision can be safely and effectively used in salvaging leaks early after pancreas transplantation. Early recognition of the complication and swift salvage are paramount to success. Further case reports and series will be helpful in establishing the reliability and reproducibility of this technique.


Journal of Experimental Botany, Advance Access publication: 05 April 2018, doi: 10.1093/jxb/ery132The original online version of this paper contained an incomplete reference, omitting the name of a lead author. The reference has been updated to read as follows:Silva GFF, Silva EM, Azevedo MDS, Guivin MAC, Ramiro DA, Figueiredo CR, Carrer H, Peres LEP, Nogueira FTS. 2014. microRNA156-targeted SPL/SBP box transcription factors regulate tomato ovary and fruit development. The Plant Journal 78, 604–618.


This composite material is used to build highly efficient near-infrared (NIR) FAPbI3 Perovskite light-emitting diodes (PeLEDs) that exhibit a high radiance of 206.7 W/sr*m2, among the highest reported for NIR-PeLEDs, obtained at a very high current density of 1000 mA/cm2, while importantly avoiding the efficiency roll-off effect. In depth photophysical characterization allows to identify the possible role of the biopolymer in i) enhancing the radiative recombination coefficient, improving light extraction by reducing the refractive index, or ii) enhancing the effective optical absorption because of dielectric scattering at the polymer-perovskite interfaces. Our study reveals how the use of insulating matrixes for the growth of perovskites represents a step towards high power applications of PeLEDs.Herein, an insulating biopolymer is exploited to guide the controlled formation of micro/nano-structure and physical confinement of α-δ mixed phase crystalline grains of formamidinium lead iodide (FAPbI Some of the optoelectronic properties of perovskites are ideal for efficient LEDs, particularly the color-tunable emission, the easy manufacturing4, and the reduced formation of active electronic trap states6. However, some of the peculiar characteristics of three-dimensional hybrid halide perovskites such as loosely bound excitons and very high carrier mobilities, instead, hinder electroluminescence. At room temperature, excitons in hybrid halide perovskites have indeed low binding energies8, therefore PLQY increases at high excitation flux when radiative bimolecular recombination dominates. In the actual PeLEDs, radiative recombination competes with trap-mediated non-radiative decay and Auger recombination mechanisms, the latter being the principal factors responsible of the device efficiency roll-off at high current density regimes10. In general, the term “efficiency roll off” is used to describe the decreasing of the efficiency of the device at high current density regimes. It can be quantified by the critical current density, which represents the current density at which the external quantum efficiency (EQE) drops to half of its maximum value11. In perovskite LEDs, the origin of this phenomenon is still controversial, but it has been recently attributed to either non-radiative processes that reduce the luminescence efficiency or to excessive population of charge carriers passing through the device without forming electron-hole pairs12. Spatial confinement of injected carries has been proven to enhance radiative recombination at low excitation levels, then improve the performance of Pe-LEDs15. The simplest approach to promote the spatial confinement of the injected charges devises the growth of ultra-thin perovskite active layer (~20 nm), which however easily leads to inhomogeneous substrate coverage causing current leakages and shorts16. Alternatively, the confinement effect has been achieved by using low-dimensional perovskite nanocrystals or by growing multiple quantum wells mixing 2D/3D perovskites19. At high current densities, however, the increased spatial confinement tends to increase Auger recombination rates, so that strategies that boost emission of PeLEDs at low current densities end up in causing an efficiency roll-off at high currents12. Preventing this phenomenon is of paramount importance and presently only few work have tried to address it, among those the most successful strategy resulted to be the tuning of the quantum well width, in 2D/3D, leading to a reduction in the local carrier density12.Halide perovskites are currently explored as active materials in light-emitting diodes (LEDs) with very promising outcomes2CH2NH3PbI3 or FAPbI3) 3D perovskite grains21. Starch is organized into linear or branched structures defined amylose and amylopectin, respectively, composed by repeating a-D-glucopyranosyl units  and δ (non-emissive) FAPbI3 phases, a crystalline scenario that have been proven to reduce the local dielectric landscape and the material average grain size, thus increasing the exciton binding energy and the spatially confinement of the excitons24, eventually leading to PLQY enhancement and to blue shift of the emission peak upon starch addition with respect to the reference. The presence of an insulating polymer, in addition, improves light extraction by reducing the refractive index or even enhancing the effective optical absorption by screening the charge at the polymer-perovskite interfaces. Finally, the better coverage of the substrate obtained with polymer improves the active interfaces leading to high current densities.Near-infrared LEDs, exploiting the peculiar prerogatives of perovskite FAPbI26. To promoted the formation of stabilized emissive α phase (from δ phase) at low annealing temperature, together with much enhanced NIR emission24 and resistance to humidity we introduce here an excess amount of FAI in the perovskite precursors (FAI: PbI2 = 2:1). FAPbI3 films were obtained by a single spin coating step of perovskite formulations with different starch biopolymer content onto polyethylenimine ethoxylated (PEIE) modified ZnO substrates. Film morphology was analyzed by scanning electron microscopy (SEM) and depicted in Figs 3 perovskite  that forms a highly symmetrical perovskite structure with a band gap of 1.48 eVite Fig. , resulti3 film in a simple single step deposition20. The hydrogen chemical interaction with the –OH groups of the starch macromolecule20 can indeed interfere with the self-aggregation properties and the complex dynamic equilibria existing in solution between the perovskite precursors, profoundly affecting the morphology of the resulting polycrystalline film. Accordingly, we found that the size of the perovskite grains is strictly related to the concentration of starch, with the crystal size decreasing by increasing the starch content, as shown in Fig. Remarkably, the physical/chemical interaction between starch Fig.  and pero3 film (red curve), showed a diffraction peak at 2θ = 13.9°(−110) and less intense diffraction peaks at 2θ = 28.1°(220) and 2θ = 31.5°(222), which corresponds to FAPbI3 α-phase28, confirming that the excess FAI contributed to the formation of α-FAPbI3 at mild annealing temperature (100 °C). The presence of 5% and 10% of starch into FAPbI3 leads to reduced intensity and broadening of the XRD peaks associated to the α-phase perovskite , corresponding to δ-phase of FAPbI326. In general, the variation of FAI precursors concentration affects the equilibrium between α and δ FAPbI3 phases, stabilizing one or the other, by acting on the colloid particle size and thus on the total free energy (including surface and bulk) of the two phases24. We found that the introduction of biopolymer led to the formation of a α-δ FAPbI3 mixed phase and the intensity of δ-phase peak was enhanced by increasing starch concentration, eventually resulting in well-defined δ-phase peaks at 2θ = 11.8° and 2θ = 26.1° and an almost total disappearance of the α-phase peak.The crystalline structure of pristine perovskite and with the addition of starch at different concentrations was examined by X-ray diffraction (XRD), as reported in Fig. It is worth to underline that, because the thicknesses of the active perovskite films are very low, especially for the 5% and 10% being ~60 nm and ~80 nm thick respectively, the XRD signals are weak and the contribution of the substrate is remarkable. In Figure 3 phases organized in grains with a reduced size homogeneously distributed on the substrate29. From FT-IR analysis  already in the precursor solution29. This can promotes the formation of an increasing amount of δ-phase in FAPbI3, by incremental addition of starch.Differential scanning calorimetry (DSC) measurements Fig.  showed tThe combined effect of the phase mixing, along with the physical confinement of such crystallite into a polymer matrix, had a dramatic effect on the performances of PeLEDs based on these composites.3 films were integrated into devices consisting of multilayer stack, as schematically depicted in the inset of Fig. 13. The normalized electroluminescence (EL) spectra of the devices (recorded at maximum EQE voltages) are reported in Fig. 3 perovskites13, blue shifted by about 14 nm, 18 nm and 50 nm after adding 5%, 10% and 15% of starch respectively. This blueshift is a first clear indication of the perturbation caused by the starch mixture to the optical properties of the perovskite films. Such a blue shift can be correlated to the α/δ phase junction, causing a change in the dielectric constant of the media surrounding the emitting crystals. In our case, the biopolymer, thanks to its insulating nature, also contributes to dielectric landscape modification. The shape of EL spectra was not altered by the starch addition nor by different bias voltages applied up to reached max EQE. Only at high voltages the emission peak blueshifted by 6 nm, 5 nm and 4 nm in presence of 0%, 5% and 10% of starch respectively, while it stayed unaltered at the higher starch concentration (15%), as reported in Fig. The FAPbI−2 and a radiance of 86 W m−2 sr−1. Noticeably the LED device containing 5% of starch reported in Fig. −2, but it reached an outstanding high radiance of around 206 W m−2 sr−1 at 4 V and at very high current density of 1020 mA cm−2. In panel b of Fig. −2, highlighting an excellent tolerance to high carrier densities regimes and a reduced efficiency roll off if compared to previous reports12. This is a very important result, as for most LEDs, the efficiencies diminished abruptly at high current densities, here, the presence of the starch allows keeping high-efficiency operation even at very high current levels. In addition, a very low turn-on voltage of 1.4 V was observed for the best device (starch 5%), even lower than the emission photon energy of 1.6 eV, suggesting thereby efficient and barrier-free charge injection into the perovskite emitter, as consequence of the improved interfaces. This, combined with the rapid increase of both current density and radiance, by orders of magnitude after turn-on, demonstrated a very low series resistance and efficient operations of the PeLED device13 as a clear evidence of the existence of a very good carrier percolation path through perovskite-interconnected domains among the insulating starch matrix and δ phase. The very high radiance would also depend from these considerations, as an easy injection and a smoother homogeneous film would allow reaching higher current withstanding no leakages nor shorts6. The absence of perturbation in the EL spectra (absence of blueshift in the sample containing 15% of starch), under incremental applied voltages, is attributed to an effective screening of the emitting crystals in relation to the electric field increase. Noticeably, the performances, in terms of radiances, currents and EQE, are among the highest reported in literature for PeLED device operating at near-infrared wavelengths, as showed in Figs S739.The maximum EQEs and radiance of LED devices as a function of starch concentration, obtained by taking the maximum value for each LED devices and by calculating the standard deviation for each formulation, are reported in Fig. −2 sr−1 at 4 V and 936 mA cm−2 of current density. However, the LED device containing 15% of starch showed a drastic reduction of performances down to 1.5% of EQE achieved at 2 V with a current density of 6.5 mA cm−2 and a radiance of 1.4 W m−2 sr−1 at 3.2 V and 33 mA cm−2. The highest starch concentration (15%) still brings the positive crystals size reduction and an improved coverage of the substrates, as seen in SEM analysis , k2 is the bimolecular (electron–hole) recombination rate constant, and k3 is the third-order Auger rate constant. In the case of free carriers and a bulk perovskite, only the bimolecular recombination is associated to emission of a photon, therefore for light-emitting applications it is of paramount importance to reduce the impact of the trap mediated and of the Auger deactivation processes. Spatial confinement increases the local carrier density and therefore radiative recombination; in the extreme case of quantum confinement, when the dimensions of the perovskite grains decrease below the Bohr radius associated with electrons and holes, typically 10 nm, electrons and holes do not move independently anymore and the radiative recombination rate becomes monomolecular, typically improving emission efficiency at low carrier densities.Aiming at a further rationalize the starch influence on the material and device performances, we investigated the optical properties of the films. In perovskite materials, a range of different charge-carrier recombination mechanisms occur, which depend on the charge-carrier density (n) to a different degree. The rate of change in n can be expressed through the following equation :1\docume3 films, as a function of starch content; the spectra are showed in Fig. 3 is located at 813 nm, in line with what reported for α-phase of this material24. The emission peak blue shifted by around 15 nm, 30 nm and 37 nm by adding 5%, 10% and 15% of starch, respectively. This observation confirmed the EL results and further underlined an effect related to the biopolymer interaction with perovskite formation. The emission quantum yield measures the efficiency of radiative recombination with respect to competing processes and, since in perovskite radiative recombination is bimolecular, its value tends to increase with carrier density. Figure Photoluminescence (PL) measurements provided useful information to understand the effect of starch addition on radiative recombination processes. The steady-state PL measurements were carried out on FAPbI0, emitted in coincidence with the excitation laser pulse  the formation of α/δ mixed phase, assisted by starch, which could improve the carrier recombination in the perovskite layer avoiding losses at the interface with the hole/electron transporting layers42; i) improved substrate coverage of the perovskite film, which hinders current leakage; iii) modification of dielectric landscape at the starch-perovskite and at the α-δ interfaces, eventually enhancing the electron-hole radiative recombination or light out diffusion.Combining morphological, structural and photophysical characterization, the overall picture that emerged is that the efficiency roll off is reduced and radiance is enhanced with starch thanks to a combiation of factors: 3 perovskite crystals into a biopolymeric matrix we achieved control on the grain sizes and on the peculiar crystalline composition. A mixed α/δ phase junction, characterized by enhanced NIR emission, reduced grains size and α phase stabilization (from δ phase) at low annealing temperature, was in fact formed into the starch network. The use of starch controls the perovskite grain size, their electronic screening at high-carrier densities as well as the substrate coverage, ensuring high performing PeLEDs. The composites embedded in LED lead to maximum radiances above 200 Wm−2 sr−1 and very high efficiencies ~5% EQE obtained at high current densitiy regimes for a minimized efficiency roll-off effect.In summary, by growing FAPbI2 ultradry 99.999%  was purchased from Alfa Aesar, Formamidinium iodide HC(NH2)2I (FAI) from Xi’an Polymer Light Technology Corp. Corn starch (Maizena) was purchased from Unilever, DMSO was purchased from Fisher BioReagents. 2-isopropanol , ethanol , Polyethylenimine (PEIE) 80% ethoxylated solution, Zinc acetate dihydrate (99.999%), were all purchased from Sigma Aldrich.Lead (II) iodide PbI33. ITO substrates were purchased from Shenzhen Huayu Union Technology Co. Ltd.Poly[-co-diphenylamine)] (TFB) was supplied from American Dye source Inc. M-xylene 99% extra pure was purchased from Acros Organics. All the materials were used as received without any further purification. ZnO nanocrystals was prepared as previous reported2 = 2:1, were prepared by mixing FAI with PbI2 in DMSO with a concentration of 10 wt% respect to the solvent. The solution was stirred at 80 °C for 30 min. After precursors solubilisation, different starch/precursors ratios were added to each solution, i.e. 0 wt%, 5 wt% 10 wt% and 15 wt%, that were stirred at 80 °C for 5 h in order to obtain a clear solution after starch solubilisation.The perovskite precursors solution with a modified precursors stoichiometry, i.e. FAI: PbIScanning electron microscope images of the starch-perovskite composite samples, deposited on ITO/ZnO-PEIE modified substrate at 7000 rpm for 100 s and annealed at 100 °C for 10 min, were collected by using Carl Zeiss Auriga40 Crossbeam instrument, in high vacuum and high-resolution acquisition mode, equipped with Gemini column and an integrated high efficiency in-lens detector. The applied acceleration voltage was 5 kV.3 film containing different starch concentrations, deposited on ITO/ZnO-PEIE modified substrate at 7000 rpm for 100 s and annealed at 100 °C for 10 min, were collected on a PW 1729 Philips, using Cu Kα radiation in reflection mode (λ = 0.154 nm). All the samples were step-scanned at room temperature from 2θ values of 5°− 40°.Wide-Angle X-ray diffraction spectra of FAPbI−1 scan rate under nitrogen atmosphere flow at 60 mL min−1. The endotherm related to solvent evaporation has been subtracted in order to isolate the perovskite crystallization exotherm.Dynamic DSC scans were performed starch-perovskite precursor solutions, without and with 5 wt% of starch/precursors ratio, by a differential scanning calorimeter (DSC Mettler Toledo 622). About 10 μL of liquid samples was put into opened aluminium flat disks and heated from 25 up to 200 °C at 10 °C min2, using a Fourier Transform Infrared spectrometer (FTIR) Jasco 6300. Each measurement was obtained with 128 scans and 4 cm2 of resolution.Infrared spectra were recorded on starch film and FAPbI3 film with and without starch, deposited on silicon substrate at 7000 rpm for 100 s and annealed at 100 °C for 10 s. The analyses were conducted in the wavelength range between 4,000 and 700 cm3: H2O2: H2O = 1:1:5) procedure for 20 min. ZnO nanoparticles were deposited in ambient condition by spin coating at 4000 rpm for 40 s and annealed at 150 °C for 30 mins. PEIE dispersed in IPA solution with a concentration of 0.05 wt% was spin coated on the top of ZnO at 5000 rpm for 40 s. Perovskite based solutions, with and without starch, were spin-coated on ITO/ZnO/PEIE in glovebox at 7000 rpm for 100 s and then the films were annealed at 100 °C for 10 min. TFB solution (8 mg/ml in m-xylene) was spin-coated onto the perovskite film. Finally, 10 nm of MoO3 film and 100 nm of Ag film were deposited as electrode inside of the thermal evaporator.ITO coated glass substrates were treated with detergent overnight and then by TL1  were used to collect emission data. The device working area is 0.0725 cm4 CW laser (Spectra Physics Millennia V) at 532 nm. The photoluminescence was dispersed with a grating spectrometer  and detected by a LN-cooled CCD camera (Princeton Instruments). The absolute photoluminescence quantum yield was calibrated using an Edinburgh FLS920 spectrometer equipped with a Peltier-cooled Hamamatsu R928 photomultiplier tube (185–850 nm). An Edinburgh Xe900 450 W xenon arc lamp was used as exciting light source. Corrected spectra were obtained via a calibration curve supplied with the instrument. Lamp power in the QY experiments about 0.6 mWcm−2, spot area 0.5 cm2.In order to measure continuous-wave photoluminescence spectra, samples were excited with a diode-pumped Nd:YVOet al.43.QY have been determined by using a barium sulphate coated integrating sphere (4 or 6 inches), following the procedure described by de Mello In time-resolved photoluminescence experiments, samples were excited with a regenerative amplified laser (Coherent Libra) delivering 130-fs-long pulses at a repetition rate of 1 KHz. Photoluminescence was dispersed with a grating spectrometer (Princeton Instruments Acton SpectraPro 2300i equipped with a 50 gr/mm grating blazed at 600 nm), dispersed and detected by a streak camera (Hamamatsu).Supplementary Information


We sought to investigate the longitudinal trajectories of the new generation epigenetic clock′s and how medication use is altering the DNA methylation age (DNAmAge). DNA methylation (Illumina 450k and EPIC) was assessed repeatedly up to six times (1992-2014) in whole blood  from the Swedish Adoption/Twin Study of Aging (SATSA). DNAmAges were generated with the online calculator. Mean age at first measurement was 67 years (58% women). All clocks tested  were correlated with chronological age (ρ=0.62-0.80). The steepest slope was found for Pheno while Horvath had the least steep slope. Correlations between the clocks ranged ρ=0.43-0.75. About 15% of the individuals started statin treatment during the follow-up, which changed the slopes to be less steep. Co-twin control analyses were confirmatory. Different DNAmAges are strongly correlated with each other in a longitudinal perspective. Treatment effects may alter the slopes of the DNAmAges.


Xenopus brain, and retinal ganglion cell axons turned to follow this gradient. Changes in local tissue stiffness were largely governed by cell proliferation, as perturbation of mitosis diminished both the stiffness gradient and the caudal turn of axons found in control brains. Hence, we identified a close relationship between the dynamics of tissue mechanics and developmental processes, underpinning the importance of time-resolved stiffness measurements.Tissue mechanics is important for development; however, the spatio-temporal dynamics of in vivo tissue stiffness is still poorly understood. We here developed tiv-AFM, combining time-lapse in vivo atomic force microscopy with upright fluorescence imaging of embryonic tissue, to show that during development local tissue stiffness changes significantly within tens of minutes. Within this time frame, a stiffness gradient arose in the developing  Neurons in the brain form an intricate network that follows a precise template. For example, in a young frog embryo, the neurons from the eyes send out thin structures, called axons, which navigate along a well-defined path and eventually connect with the visual centres of the brain. This journey requires the axons to take a sharp turn so they can wire with the right brain structures.Axons find their paths not only by following chemical signals but also by reacting to the stiffness of their environment. In an older frog embryo for instance, the brain is stiffer at the front, and softer at the back. As neurons from the eyes make their way through the brain, they turn to follow this gradient, moving away from stiffer areas towards the softer regions.Here, Thompson, Pillai et al. investigate when and how this stiffness gradient is established in frogs. To do so, a new technique was developed. Called time-lapse in vivo atomic force microscopy, the method measures how brain stiffness changes over time in a live embryo, while also taking images of the growing axons.The experiments show that the stiffness gradient arose within tens of minutes, just as the first ‘pioneering’ axons from the eyes began to grow across the brain. These axons then responded to the gradient, turning towards the softer tissue. Changes in the number of cells in the underlying brain tissue governed the formation of the gradient, with rapidly stiffening areas containing more cells than those that remained soft. In fact, using drugs that stop cells from dividing reduced both the mechanical gradient and the turning response of the axons.The technique developed by Thompson, Pillai et al. is a useful tool that can help elucidate how variations in stiffness control the brain wiring process. It could also be used to look into how other developmental or regenerative processes, such as the way neurons reconnect after injuries to the brain or spinal cord, may be regulated by mechanical tissue properties. During embryonic development, many biological processes are regulated by tissue mechanics, including cell migration , neuronaTo enable time-resolved measurements of developmental tissue mechanics, we here developed time-lapse in vivo atomic force microscopy (tiv-AFM), a method that combines sensitive upright epifluorescence imaging of opaque samples, such as frog embryos, with iterated AFM indentation measurements of in vivo tissue at cellular resolution and at a time scale of tens of minutes . A fluorXenopus embryo brain during outgrowth of the optic tract (OT) as a model  and cantilever probe (r = 18.64 µm) were chosen to measure the stiffness mainly of the top ~20–30 µm of the tissue, within which RGC axons grow , OTs were labelled with DiI , the stiffness of the brain was similar on both sides of the OT. However, over the time course of the measurements a stiffness gradient arose, mostly due to rising stiffness of tissue rostral to the OT  correlate with a gradient in cell density -positive cells , which inhibits DNA replication  and has  embryos . HUA als embryos .G’~5,500 Pa was not altered by the presence of BI2536 if compared to control conditions  at 14–18°C to reach the desired developmental stage, as described by All animal experiments were approved by the Ethical Review Committee of the University of Cambridge and complied with guidelines set by the UK Home Office. Single-cell-stage, wild-type ath5::GAP-eGFP construct (pCS2+ vector)   was injepromoter . This seStage 33/34 embryos were anaesthetised, the eye primordium was removed and the underlying brain hemisphere exposed as described . BrieflyBI2536 inhibitor or mock-treated embryos were fixed at the requisite stages in 4% PFA overnight at 4°C, washed thrice in phosphate-buffered saline (PBS) for 10 min, and kept in 30% sucrose for 1 hr at 4°C. The embryos were embedded in optimum cutting temperature compound . 12µm-thick coronal sections were made and collected on Superfrost plus slides (ThermoScientific).Polyacrylamide hydrogels were prepared as previously described . BrieflyG (a measure of stiffness) of ~300 Pa . For gels used for stiffness sensing experiments, the precise stiffness was measured using AFM. Stiff gels were comprised of 7.5% AA/0.2% Bis-AA in PBS, resulting in a shear modulus of G ~5,500 Pa; soft gels were comprised of 5% AA/0.04% Bis-AA in PBS, resulting in G ~200 Pa.Gel pre-mixes were prepared using 40% (w/v) acrylamide (AA) solution and 2% bis-acrylamide (Bis-AA) solution  diluted in PBS. Concentration titration measurements used a premix composition previously determined to give a shear modulus Premix polymerization was initiated by adding 5 µL ammonium persulfate followed by 1.5 µL of N,N,N’,N’-tetramethylethylenediamine . 25 µL of premix was pipetted onto the bottom coverslip and the top coverslip placed on top. Once the gel had polymerized, the top coverslip was removed and gels were treated with hydrazine hydrate for 3.5–4 hr, and then with 5% acetic acid (ACROS Organics) for 1 hr. Gels were then washed, sterilized by 30 min UV treatment, and functionalized with 10 µg/mL Poly-D-lysine  overnight followed by 5 µg/mL laminin for 2 hr immediately prior to plating cells.Xenopus cell culture medium . In all experiments, DMSO controls utilized the amount of DMSO equivalent to that in the most concentrated BI 2536 condition . Explants were imaged on a Leica DMi8 inverted microscope with a 10× NA = 0.4 phase contrast objective.For eye primordia culture experiments, stage 33/34 or 35/36 embryos were placed in a Petri dish coated with Sylgard 184 (Dow Corning) and anaesthetized with 0.04% (w/v) MS222 solution . Whole eye primordia were dissected out using insect pins, and placed onto hydrogels with the lens facing up. BI2536 or control solution (DMSO) was added 2 hr after explants were plated. Dishes were cultured at 20°C for 22–24 hr in R Eye primordia explant morphology was analyzed using the Sholl Analysis plugin in Fiji . An elliath5::GAP-eGFP or by injecting a solution of DiI crystals diluted in ethanol at the boundary between lens and the retina (as previously described ath5::GAP-eGFP-labelled OTs) or 1 × PBS (DiI-labelled OTs) and the lateral brain surface, including the OT, was imaged using an SP-8 confocal microscope .Where required, to visualise the OT and nuclei for cell body density measurements, embryos at the desired developmental stage were fixed in 4% paraformaldehyde for 1.5–2 hr at room temperature or overnight at 4°C. OTs were either labelled with Sectioned tissues on slides were washed thrice in PBS, followed by three 10 min washes in PBS with 0.1% TritonX. The sections were blocked in 5% goat serum in PBS with 0.1% TritonX for 30–45 min and incubated with Rabbit polyclonal anti-phospho-Histone H3 (Ser10)  overnight at 4°C or for 2 hr at room temperature. This was followed by three 10 min washes in PBS and secondary antibody incubation with goat anti-rabbit Alexa Fluor 594  for 45–60 min. The slides were washed twice for 10 min with PBS and nuclei were labeled using 4,6-diamidino-2-phenylindole . Sections were mounted with Fluoromount-G (eBioscience) and imaged with a confocal microscope . Z-stacks were taken across 8 µm of tissue (z-step size = 1 µm). Only slices with eye tissue present were selected, to reliably ensure that the brain sections imaged and analysed were indeed exposed to the treatment solutions. This also allowed us to easily ascertain the intact- versus exposed- brain side in each section.k, and those with k between 0.02–0.04 N/m were selected. Monodisperse spherical polystyrene beads  were glued to the cantilever ends as probes. Cantilevers were mounted on a CellHesion-200 AFM head (JPK Instruments), which was set up on an x/y motorised stage (JPK Instruments) controlled by custom-written Python scripts  were calibrated using the thermal noise method  to deter scripts . Indenta scripts .Xenopus brains, which are optically opaque, an upright epifluorescence set-up was custom-fit above the AFM head, which was based on a Zeiss AxioZoom V16 fluorescence stereomicroscope (without eyepieces). The microscope was mounted on a custom-designed horizontal sliding stand (parts obtained from ThorLabs) via a custom-made adaptor (K-Tec Microscope Services) bolted to the side of the microscope  were selected. Embryos were mounted on the AFM motorised stage and a measurement region of approximately 150 × 250 µm defined to include both the growing OT and the region of the mid-diencephalic turn. Images of the upper right and lower left corners of the selected region (with cantilever approached) were collected to identify the precise area mapped by the AFM. A stiffness map of the area was collected and the map iterated over the same area every ~35 min. At the end of every line in the measurement grid, the stage was moved back to a pre-defined location and a fluorescence image of the optic tract automatically collected. Temperature was maintained at 25°C for the duration of the measurement by a PetriDish Heater (JPK Instruments).Stage 33/34 embryos with K. Raw AFM data were fitted to the Hertz model,F is the applied force, K the reduced apparent elastic modulus E/(12-ν) (with Poisson ratio ν), R the radius of the indenter, and F = 10 nN. Points where the AFM data were not analysable were excluded; criteria for excluding individual force-distance curves were (1) inability to apply linear fits through the baseline region, for example due to noise, and (2) inability to apply good-quality Hertzian fits to the indentation region, that is, the fit did not align with the raw data.Force-distance curves obtained from stiffness measurements were analysed with a custom-written MATLAB script  to obtaientation . Force-dx-, y-, and time dimensions using an algorithm based on the discrete cosine transform by Garcia et al (x*y*frame number) and used as an argument for the smoothn.m MATLAB implementation of the algorithm  on the rostral (RK) and caudal sides (CK) of the OT, and the gradient calculated by: Another MATLAB script  was usedFor each frame in a given time-lapse series, ROIs were defined three times and the mean of the three measurements for each ROI was used for further analysis. Where direct comparison between the dynamics of stiffness gradients and OT turning was required across different animals, values for both parameters were rescaled. The minimum value obtained for each embryo measured was set to 0 and the maximum set to 1, using the following formula:When linear fits to AFM data and OT turning angles were required, the built-in MATLAB first degree polynomial fit was used in all cases.For each embryo, in vivo time-lapse movies of the growing OT were created by collating fluorescence images taken at the beginning of every successive AFM stiffness map. The magnitude of the OT turn was calculated from each frame of these movies using the FIJI Angle tool. Three points were defined manually  such that the two lines drawn through these points ran through the centre of the OT. Each measurement was repeated three times, averaged, and subtracted from 180° to give the angle through which the OT had turned; a positive value denotes turning in the caudal direction.Maximum projections were made across 10 µm confocal image stacks of wholemount brains with DiI-labelled OTs. The OTs were manually outlined in Adobe Illustrator. Elongation of the OT was calculated by the major-to-minor axis ratio using a previously described automated algorithm in Matlab . In shorRD) and caudal (CD) to the region corresponding to the OT caudal bend. Noise was removed with a Gaussian blur filter (sigma = 2.0). The resulting image was thresholded, with the threshold manually adjusted to capture all nuclei as accurately as possible. The image was binarised and the FIJI built-in function ‘Analyse Particle’  was used to determine the area in each region covered by nuclei. Relative cell body density was calculated by dividing the area occupied by nuclei by the total region area. Where required, gradients in relative cell body density were obtained using:Confocal image stacks of fixed brains (with fluorescently-labelled OT and nuclei) were imported into FIJI. For each brain, the image where the leading axons were in focus was selected. A maximum intensity projection was generated of this image and one image above and below it in the stack. Two 50 μm × 50 μm regions of interest  were selected, rostral . The brain was identified in each section and both intact and exposed hemispheres were outlined. The area occupied by nuclei in each brain hemisphere was measured using the ‘Analyse > Measure’ tool. Normalised brain areas were obtained by calculating the ratio of exposed brain area to intact brain area.2 of brain tissue.Confocal images taken with the 20 × air objective were imported into FIJI. Maximum projections were made of the stacks (z-stack height = 8 µm) and pH3+ cells were counted and normalized to the total measured brain area in each section. The data is presented as the number of pH3+ cells per 10,000 µm2 values provide an estimate of the quality of the fits used in the plots. Pearson’s correlation coefficient, on the other hand, provides a measure of the magnitude of correlation between the cell body density gradient and the stiffness gradient.Data were collected from at least three independent experiments (N ≥ 3). The order of data collection was randomized with no blinding and no data were excluded from the analysis. Non-parametric tests as well as linear regression analysis were used for statistical analyses of the data as described in the figure captions. R In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Marianne Bronner as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Paola Bovolenta (Reviewer #1); Valerie Castellani (Reviewer #2).Thank you for submitting your article "Rapid changes in tissue mechanics regulate cell behaviour in the developing embryonic brain" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Xenopus brain is influenced by relatively rapid changes in tissue stiffness on which the axons grow, and that increasing stiffness correlates with enhanced cell density/proliferation, are indeed novel. Your approach of simultaneously observing and quantifying tissue stiffness and cell movements using atomic force microscopy with fluorescence imaging is exciting and will appeal to the growing number of groups interested in tissue morphogenesis.The three reviewers appreciated your efforts to probe how dynamic changes in tissue mechanics have a close relationship with developmental processes, here, with a focus on axonal growth. Your findings that the pathway that retinal ganglion cell axons follow in the Essential revisions:These experiments are considered necessary to strengthen the connection you make between proliferation, density, and tissue stiffness:1) Coronal sections of control and BI2536 treated embryos showing nuclei (DAPI stained) and glial endfeet (GFAP or Nestin labelling).2) Labelling sections with pH3 or other mitotic cell markers.3) Effects of the BI2536 inhibitor: Additional movies are needed .4) Check whether RGC axons grow and sense stiffness normally in vitro in presence of BI2536 .5) Calculate optic tract (OT) elongation in control and BI2536-treated conditions. Related to BI2536 treatment and turning in the optic tract: does BI2536 generally affect axon growth along the optic tract in vivo?6) Demonstrate that repeated AFM measurements do not affect OT turning (by comparing with movies without AFM measures).7) Confirm with a second drug that cell density controls brain stiffness and RGC axon turning.The following points do not rely on additional experiments but should be addressed:1) State in the text the optical resolution and the accuracy of the AFM measure relative to the surface distance .2) Provide illustrations of the angle measurements .3) Show the ROIs used to quantify the stiffness gradient and the boxplot of the mean K values in rostral and caudal regions at different time points. In addition, using different ROIs along the OT would strengthen the specificity of the correlation between stiffness gradient and turning.4) Show several examples of the stiffness changes to better appreciate the spatial stereotypy relative to OT development.5) Figure 1D, related to text "a stiffness gradient arose, mostly due to rising stiffness of tissue rostral to the OT". Some of the heatmap data  suggests that there is decreasing stiffness on the caudal side, and relative cell density data  suggests that there is decreasing cell density on the caudal side as well. Can the authors comment on the rostral-caudal stiffness v cell density? Is there some underlying biology to account for this?6) Explain why only 3 time points are presented in the graph Figure 2A and G even though measurements are made every 35 minutes.7) Figure 2B: According to the graph, it appears that the stiffness gradient is projected to form at 8-10 minutes. This suggests that it may begin to form prior to the beginning of the experiments presented (if the gradient forms linearly at those earlier times). Have the authors tried starting the experiments earlier? The manner in which the stiffness gradient forms (linearly or non-linearly) may have implications for the mechanism(s) initiating and influencing stiffness.8) Specify the estimated delay between gradient stiffness establishment and OT caudal turn (15-18 minutes). Paired analysis of the onset times of stiffness gradient and turn for each video (ladder graphic) would strengthen the arguments. It would be interesting to have an estimate of the stiffness differences that would lead to axon turning. This would be helpful together with point 4 to make predictions and to enrich the model proposed in Koser et al., 2016.9) Show a 3D reconstruction of the Z-stack used for Figure 2C and specify the total stack height.10) Explain how "cell body density gradients" were calculated  . The authors should take into account that quantification made on a Z projection will lead to loss of information that will decrease the sensitivity of the analysis. Figure 2E suggests that a more balanced conclusion should be drawn from the experiment, as part of gradient stiffness is independent of cell density gradient (gradient stiffness would thus be 0.4 for no cell density gradient).11) Provide relative cell body density for the BI2536 condition, as done in Figure 2D for the control ").12) The Materials and methods section indicates that relative density involves ratio of 2D area in the image occupied by nuclei over total area (this does not appear to be a cell count). This measurement could be affected by differences in nuclear size. Have the authors measured nuclear size over time? Does this change? Is nuclear size different in BI2536-treated embryos? Also, it is difficult to see nuclei on the caudal side of the optic tract in Figure 2C (control).13) Again, related BI2536 and cell density measurements, over time for BI2536-treated embryos: It would be useful to know if the BI2536 caused apoptosis ? The nuclei look somewhat condensed on what appears to be the rostral side of the tract in the BI2536 sample by st. 37 .14) Show a different control movie in Figure 2F .15) Justify the changes of presentation between Figure 2A and G.16) Explain what BI2536 targets (inhibitor of polo-like kinase).2= 0.95 in Figure 2C and Pearsons coefficient in the text (p=0.97). Add scale bars in Figure 2C and F.17) Explain differences between coefficient RFor consideration in the Discussion:1) You suggest that the "gradient in cell densities is responsible for the stiffness gradient". However, the opposite could be true, as changes in stiffness have been shown to promote proliferation (should be referenced). In addition, as you previously showed that ECM manipulation changes the tissue stiffness and axon trajectory , it would be welcome to discuss whether you have assessed whether changes in cell density correlate with changes in ECM deposit and if BI2536 impairs it.2) In the last paragraph, you discuss applications of tiv-AFM to other possible contexts. Limitations of the system should be also mentioned. Essential revisions:These experiments are considered necessary to strengthen the connection you make between proliferation, density, and tissue stiffness:1) Coronal sections of control and BI2536 treated embryos showing nuclei (DAPI stained) and glial endfeet (GFAP or Nestin labelling).Xenopus brains at the early developmental stages we were interested in . Furthermore, we could not find a Nestin antibody with sequence similarities above 85% with Xenopus . We also stained for vimentin, which is another glial cell marker. While we obtained a good signal from that approach, we could not distinguish individual glial cell endfeet. As we cannot make any strong conclusions about the contribution of glial cells to the stiffness gradients and axon turning based on these data . However, GFAP is not expressed in data see , we now 2) Labelling sections with pH3 or other mitotic cell markers.We have now labelled coronal sections with an antibody to detect histone H3 phosphorylated at serine 10, a marker of mitotic cells. Ser10 phosphorylation begins in G2 and decreases upon exit from mitosis. We found that the BI2536 treatment successfully triggers mitotic arrest as the number of pH3-positive cells is significantly higher in treated compared to control brains. These data have been added to Figure 4A, C.3) Effects of the BI2536 inhibitor: Additional movies are needed .We have repeated these experiments with 2 more biological replicates and updated the numbers in the paper (N = 4). The new sets of experiments fully agree with our previous data, confirming reproducibility of the experiments and that the stiffness gradients is almost completely abolished after BI2536 treatment .4) Check whether RGC axons grow and sense stiffness normally in vitro in presence of BI2536 .We have done the suggested in vitroexperiments. Axons exposed to 50nM and 500nM BI2536 still sense and respond to the stiffness of their environment, suggesting that the main effect of the drug is through a modification of tissue mechanics. The new data have been added to Figure 4—figure supplement 3.5) Calculate optic tract (OT) elongation in control and BI2536-treated conditions. Related to BI2536 treatment and turning in the optic tract: does BI2536 generally affect axon growth along the optic tract in vivo?We have calculated OT elongation as suggested and find differences between controls and BI2536-ând HUA-treated animals. In the treated animals, elongation is significantly reduced, which we now show in Figure 4G and Figure 4—figure supplement 2D. As brain tissue stiffness is decreased in treated animals with decreased nuclear densities , which is in agreement with an important role of cell body density in regulating tissue stiffness, a reduction in OT elongation is in line with our previous publication .6) Demonstrate that repeated AFM measurements do not affect OT turning (by comparing with movies without AFM measures).To address this point, we conducted time-lapse AFM measurements (i.e. repeated AFM mapping over a time course of at least 3 hours) on a group of embryos while we exposed another group to the same conditions but did not do any force measurements. At the end of the experiments , embryos were fixed, optic tracts labelled with DiI, and their elongation and turning angles measured. We did not find any significant differences between the groups, suggesting that repeated AFM measurements do not affect OT turning. We have added the new data to Figure 2—figure supplement 2 and the fifth paragraph of the main text.7) Confirm with a second drug that cell density controls brain stiffness and RGC axon turning.Xenopus embyros in vivo: Harris and Hartenstein, 1991), which resulted in a very similar optic tract phenotype as seen in BI2536-treated brains, with significantly reduced OT elongation and brain stiffness. These experiments are now shown in Figure 4—figure supplement 2.We have now used a second mitotic inhibitor treatment, hydroxyurea/aphidicolin (HUA)  State in the text the optical resolution and the accuracy of the AFM measure relative to the surface distance .AFM is applied to the surface of a sample. The measured stiffness is a composite property of structures within the top layers of the sample, with a larger contribution of structures that are closer to the surface. The total depth that contributes to the measured value depends on the mechanical properties of the sample, the force applied, and the contact area between probe and sample. In our experiments, we indent the samples by ~3-4 µm. We estimate structures to contribute to the measured stiffness mainly within the top ~20-30 µm (see simulation in 2) Provide illustrations of the angle measurements .Done as suggested .3) Show the ROIs used to quantify the stiffness gradient and the boxplot of the mean K values in rostral and caudal regions at different time points. In addition, using different ROIs along the OT would strengthen the specificity of the correlation between stiffness gradient and turning.K values in rostral and caudal regions at different time points in Figure 2—figure supplement 1. However, we have chosen the ROIs to be directly in front of the advancing OT axons because this is the region that growth cones will be sensing. Therefore, we would rather not include different ROIs along the OT to avoid confusion, as it is not clear whether these would contribute to axon guidance in any way.We now show the ROIs used to quantify the stiffness gradient and boxplots of the median 4) Show several examples of the stiffness changes to better appreciate the spatial stereotypy relative to OT development.We now show two more examples of time-lapse stiffness maps in Figure 2—figure supplement 3.5) Figure 1D, related to text "a stiffness gradient arose, mostly due to rising stiffness of tissue rostral to the OT". Some of the heatmap data  suggests that there is decreasing stiffness on the caudal side, and relative cell density data  suggests that there is decreasing cell density on the caudal side as well. Can the authors comment on the rostral-caudal stiffness v cell density? Is there some underlying biology to account for this?We generally see a strong correlation between local cell body densities and tissue stiffness. Accordingly, tissue stiffness increases rostral to the OT, where cell density increases during development. Caudal to the OT, cell density decreases slightly in some but not all embryos, and accordingly we don’t see statistically significant differences in tissue stiffness caudal to the OT.6) Explain why only 3 time points are presented in the graph Figure 2A and G even though measurements are made every 35 minutes.Xenopus embryos encompassing the duration of our experiments , in order to enable meaningful comparisons with local cell body densities, which were assessed at these stages. Therefore, values from stiffness maps had to be pooled. We now justify this procedure in the caption of Figure 2.Our aim was to match the mechanics measurements to the three established developmental stages of 7) Figure 2B: According to the graph, it appears that the stiffness gradient is projected to form at 8-10 minutes. This suggests that it may begin to form prior to the beginning of the experiments presented (if the gradient forms linearly at those earlier times). Have the authors tried starting the experiments earlier? The manner in which the stiffness gradient forms (linearly or non-linearly) may have implications for the mechanism(s) initiating and influencing stiffness.Our data indeed suggest that stiffness gradients may start forming a few minutes before the start of our measurements. However, our data strongly indicate that the stiffness gradient forms rather linearly during the following ~3 hours between stage 33/34, when the pioneering axons first leave the optic chiasm and enter the diencephalon, and stage 37/38, when axons have turned in the mid-diencephalon . Measurements at earlier developmental stages would be technically highly challenging.8) Specify the estimated delay between gradient stiffness establishment and OT caudal turn (15-18 minutes). Paired analysis of the onset times of stiffness gradient and turn for each video (ladder graphic) would strengthen the arguments. It would be interesting to have an estimate of the stiffness differences that would lead to axon turning. This would be helpful together with point 4 to make predictions and to enrich the model proposed in Koser et al., 2016.These are indeed excellent suggestions. We now specify the delay of 18 minutes in the seventh paragraph of the main text and in Figure 2E. Furthermore, we include a ladder graphic as suggested, in addition to our paired analysis of the onset times, in Figure 2D. We now also include an estimate of the minimum stiffness gradient that would lead to axon turning, which is (0.9 ± 0.4) Pa/µm.9) Show a 3D reconstruction of the Z-stack used for Figure 2C and specify the total stack height.As mentioned in the Materials and methods, we only recorded three optical planes in each stack to make sure we capture all nuclei in that region. Hence, a 3D reconstruction would not add to the information that is already provided. Moreover, the coronal sections that we now added  contain information about the depth-distribution of nuclei, which we hope addresses this point.10) Explain how "cell body density gradients" were calculated  . The authors should take into account that quantification made on a Z projection will lead to loss of information that will decrease the sensitivity of the analysis. Figure 2E suggests that a more balanced conclusion should be drawn from the experiment, as part of gradient stiffness is independent of cell density gradient (gradient stiffness would thus be 0.4 for no cell density gradient).We apologise for this oversight and added an explanation of cell body density gradient calculations to the Materials and methods. As mentioned above, our Z projections consisted of three optical slices only that were spaced by 2 µm, so that we would expect the loss of information to be rather small.2 value is still 0.74. However, we agree with the reviewer that other tissue components might very well also contribute to setting up the stiffness gradient to some degree, and we now tone our conclusions down and mention potential other sources including radial glial cells and the extracellular matrix. We would like to point out, however, that even if other cellular and/or extracellular components are involved in setting up an initial stiffness gradient (which would not be strong enough to cause axon turning), our experiments with two different mitotic blockers strongly suggest that the cell body density gradient is responsible for the development of this stiffness gradient to the point where it impacts axon turning, as perturbations of mitosis abolish the stiffness gradient almost completely.The extrapolation of the fit in Figure 2E is an excellent point. When we force the fit through the coordinate origin (which would reflect a scenario in which the cell body density gradient is the only major contributor to the stiffness gradient), the R11) Provide relative cell body density for the BI2536 condition, as done in Figure 2D for the control ").Xenopus brain suggested that in BI2536-treated brains, the nuclear density decreased particularly rostral to the OT .’We now provide a relative cell body density count for the BI2536 condition  and rephrased our conclusion: ‘The inspection of the stage-dependent distribution of cells in the developing 12) The Materials and methods section indicates that relative density involves ratio of 2D area in the image occupied by nuclei over total area (this does not appear to be a cell count). This measurement could be affected by differences in nuclear size. Have the authors measured nuclear size over time? Does this change? Is nuclear size different in BI2536-treated embryos? Also, it is difficult to see nuclei on the caudal side of the optic tract in Figure 2C (control).The reviewer is correct, we intentionally quantified the area occupied by nuclei rather than their number. Nuclei are usually much stiffer than, for example, cytoplasm. From a mechanical point of view , it does not matter whether the total nuclear area increases because of a change in nuclear size or number. The number of nuclei caudal of the OT is indeed very low, which likely explains (at least to some degree) why the tissue in that region is softer than cranial of the OT, where a significantly larger volume is covered by nuclei.13) Again, related BI2536 and cell density measurements, over time for BI2536-treated embryos: It would be useful to know if the BI2536 caused apoptosis ? The nuclei look somewhat condensed on what appears to be the rostral side of the tract in the BI2536 sample by st. 37 .We cannot rule out that BI2536 caused some degree of apoptosis at the application site. However, again, from a mechanical point of view it does not matter much why nuclear density changes. The scope of this paper is to show that local tissue stiffness may change rather rapidly during development, and that these changes are largely governed by changes in nuclear densities. We would like to point out that retinal ganglion cells, whose axons grow along the optic tract, are located within intact eye primordia and are thus not exposed to the drug in exposed brain preparations.14) Show a different control movie in Figure 2F .We intentionally showed the same data in Figures 1 and 2, as we wanted to make a comparison easier . However, we see the value of showing different time lapse data and added two more series to the supplement .15) Justify the changes of presentation between Figure 2A and G.In Figure 2A , we show the stiffness gradients and turning angles normalised by the maximum values measured, which allowed us to extrapolate linear fits and determine the relative onset of both gradients and angles . This normalisation is very useful as absolute values will vary slightly not only between individual animals but also between different AFM cantilevers. In Figure 2G , however, we wanted to compare treated embryos with controls. If we would follow the same normalisation process, the gradients of both groups would terminate at 1 and no differences would be visible. This problem was circumvented by normalising with respect to the first measurement in a time series . This way, Figure 2G shows clearly that in controls but not in treated animals stiffness gradients and the turning angles are increasing over time.16) Explain what BI2536 targets (inhibitor of polo-like kinase).Done as suggested .2= 0.95 in Figure 2C and Pearsons coefficient in the text (p=0.97). Add scale bars in Figure 2C and F.17) Explain differences between coefficient R2 value provides an estimate of the quality of fits to data distributions. Pearson’s correlation coefficient, on the other hand, provides a measure of the magnitude of correlation between the cell body density gradient and the stiffness gradient. We have added this information to the Methods. We have now increased the visibility of the scale bars in the figures.The RFor consideration in the Discussion:1) You suggest that the "gradient in cell densities is responsible for the stiffness gradient". However, the opposite could be true, as changes in stiffness have been shown to promote proliferation (should be referenced). In addition, as you previously showed that ECM manipulation changes the tissue stiffness and axon trajectory , it would be welcome to discuss whether you have assessed whether changes in cell density correlate with changes in ECM deposit and if BI2536 impairs it.We would like to thank the reviewer for this comment. If cell density-independent changes in tissue stiffness would drive the growth of the stiffness gradient and independently lead to differential cell proliferation, interfering with cell proliferation should not diminish the stiffness gradient. However, our data show that the application of BI2536 almost completely abolishes the stiffness gradient, suggesting that it is rather unlikely that a cell density-independent stiffness gradient is responsible for the gradient in cell densities as suggested by the reviewer. However, the increase in cell density and thus tissue stiffness could provide a positive feedback signal, facilitating further cell proliferation as suggested by the reviewer. We have added this to the Discussion and provide a relevant reference.We have not assessed whether changes in cell density correlate with changes in ECM deposition and if BI2536 impairs it. At the stages investigated in this study, there is very little ECM in the brain , and we would thus not expect a major effect either due to or impacting the ECM. However, we have added a potential contribution of the ECM to tissue stiffness in the Discussion.2) In the last paragraph, you discuss applications of tiv-AFM to other possible contexts. Limitations of the system should be also mentioned.This is a good point. We now discuss explicitly limitations of tiv-AFM, which are mainly that the method is restricted to surfaces and the time each map takes, which currently limits whole-brain scans at cellular spatial resolution to a temporal resolution of about half an hour.


The maximum and minimum WAC was observed in samples containing 100% pregelatinized and 100% cross‐linked starches, respectively. An increase in the amount of pregelatinized starch was accompanied by a decrease in the maximum viscosity and gelatinization temperature. Pregelatinized starch improved sensory and textural characteristics of samples, while cross‐linked starch affected baking properties of the product. The best sample containing 30% of pregelatinized and 70% cross‐linked starch with 1.27 g protein and 12 mg phenylalanine in 100 g biscuit is introduced as the low protein and safe product for Phenylketonuria.The effect of corn starches  in four levels on quality of low‐protein biscuit was evaluated. First, the microscopic structure, thermal properties, and low‐branched chains content of them were evaluated. Water absorption capacity (WAC) and gelatinization properties of combined starches and physicochemical properties of products were assessed. Cross‐linked starch had the highest glass transition temperature ( gT). The maximum and minimum WAC was observed in samples containing 100% pre‐gelatinized and 100% cross‐linked starches, respectively. An increase in the amount of pre‐gelatinized starch was accompanied by a decrease in the maximum viscosity and gelatinization temperature. Pre‐gelatinized starch improves sensory and textural characteristics of samples, while cross‐linked starch affected baking properties of the product. The best sample containing 30% of pre‐gelatinized and 70% cross‐linked starch with 1.27 g protein and 12 mg phenylalanine in 100 g biscuit is introduced as the low protein and safe product for Phenylketonuria.The effect of corn starches  in four levels on quality of low‐protein biscuit was evaluated. Cross‐linked starch had the highest glass transition temperature ( It is due to mutations in both copies of the phenylalanine hydroxylasegene and chromosome 1, which is responsible of making this liver enzyme. Mutations in the structure of this enzyme disturb converting phenylalanine to tyrosine and as increase the accumulation of phenylalanine in blood, neurons, and brain. In people with the normal function of the enzyme, phenylalanine converts to tyrosine and other by‐products, and therefore, the amount of blood phenylalanine remains at a normal level  has profound undesirable effects on textural and qualitative characteristics of low‐protein biscuits.Due to insufficient information about effect of modified starch on qualitative properties of low‐protein cereal‐based products, in present study, first, the qualitative characteristics of three types of corn starch  and the mixture of them were investigated. Then, their effects on quality of low‐protein biscuits were evaluated to understand the relationship of qualitative characteristics of starches and products made from them.22.1Natural and modified starches (pregelatinized and cross‐linked) were provided from Glucosan Company . Tara gum was obtained from Egzandal Corbe (America), and Lecithin and mono, diglyceride were purchased from Behpak companies  and Dalian (China), respectively. Sodium bicarbonate and ammonium were obtained from Kimia acid company . Sugar and baking powder, vanilla, egg, and margarine butter were provided from local markets.2.2Thermal behavior of the starches was evaluated by Differential Scanning Calorimetry (DSC) (model DSC 200 F 3). The calibration of instrument was performed by silver and indium. Three mg of the samples was scanned in the thermal range of 30–320°C at the rate of 10°C/min . For this purpose, the samples were covered by a thin gold layer, and their appearance was investigated by an electronic microscope with an electrical potential of 5 kV  after storage at 20°C for 24 hr. The samples were fixed in 3 cm distance, and the brittleness in central part of them was investigated with a knife‐edge probe (60 mm width and 3 mm thickness) in speed of 0.8 mm/s  was employed for determining the amino acids profile (Perkin‐Elmer). 0.2 g of defatted sample using chlorophyll and methanol was hydrolyzed by HCL (6 M) for 24 hr at 110°C. HCL was removed in rotary at 45°C, and dried sample was prepared for the amino acid derivation by phenyl isothiocyanate. 1–10 µl of the samples was injected to C18 column (9.3 mm × 300 mm inner diameter). A standard solution containing amino acids  was prepared. The amino acids were identified by retention time comparison with authentic standards. Quantification was performed on the basis of the standard method  using fluorescence signals at a wavelength of 254 nm. Chromatographic data were analyzed using a Breeze Controller software   according to completely randomized design followed by LSD's test to compare the means of the dependent variables at significant probability level through the statistical software version 9 (SAS). The correlation coefficients of parameters were investigated based on Pearson test.SD). Diagrams were constructed using the Microsoft Excel 2010 software.All the measurements were carried out in triplicates, and the results were presented as mean ± standard deviation . Cross‐linking process provides stronger interaction among starch chains and consequently increases their resistance against water absorption  on product diameter  . Therefore, the samples containing 100% cross‐linked and 100% pregelatinized starch had minimum and maximum density, respectively. High water absorbability of starch reduced expansion rate of product in heating process. Therefore, maximum density was observed in the biscuit contain maximum amount of starch with the highest water absorbability. Increasing the viscosity of dough containing pregelatinized starch at ambient temperature also effectively reduced air bubbles release and increased product density during heating process   Figure .2 are an effective parameters in reducing volume of biscuit  is 10 g per 100 g of the sample. Therefore, about 85%–90% reduction in protein content of the selected sample was achieved. By considering the acceptable limit of daily proteins (50–65 g) for PKU, one serving of this produce (about 30–50 g) supplies only 1% of the daily consumable protein of patient. Peroxide index, ash content, and pH of this sample were 0.06 ± 0.02 (mg oxygen/kg of fat), 1.05 ± 0.05 (g/100 g of product) and 6.8 ± 0.3 which were in an acceptable range according to standard qualify attributes of this product .Amino acids profile of the selected sample is presented in Table 4Modification of starch had specific effects on its properties such as the granules appearance in microscopic imaging, thermal properties such as glass transition temperature and gelatinization characteristics, and water absorption capacity. Specification of starch had considerable effects on the qualitative properties of low‐protein biscuit. Cross‐linked starch reduced viscosity and induced heat stability of granules during product baking in comparison to natural starch. The best composition of starch‐based on the qualitative attributes of the products with the highest sensorial acceptability was the mixture of 70% cross‐linked and 30% pregelatinized starches. This product with 1.27 g protein  in 100 g biscuit is introduced as low‐protein biscuit and a safe product for Phenylketonuria disease. It was found that in each serving of low‐protein biscuit formulated by modified starches, 15–35 mg phenylalanine that supplies about 5%–10% of the allowable daily consummation of this amino acid for Phenylketonuria patients will be received (300–750 mg/day). Thus, a diet containing the developed biscuit can help to the care of Phenylketonuria patients.The authors declare no conflict of interest.This article does not contain any studies with human participants or animals performed by any of the authors.