Here we report the prediction of a compound, Ti2InB2, a stable boron-based ternary phase in the Ti-In-B system, using a computational structure search strategy. This predicted Ti2InB2 compound is successfully synthesized using a solid-state reaction route and its space group is confirmed as Pm2 (No. 187), which is in fact a hexagonal subgroup of P63/mmc (No. 194), the symmetry group of conventional Mn+1AXn phases. Moreover, a strategy for the synthesis of MXenes from Mn+1AXn phases is applied, and a layered boride, TiB, is obtained by the removal of the indium layer through dealloying of the parent Ti2InB2 at high temperature under a high vacuum. We theoretically demonstrate that the TiB single layer exhibits superior potential as an anode material for Li/Na ion batteries than conventional carbide MXenes such as Ti3C2.M Two-dimensional materials are promising for electrochemical storage and conversion, but are somewhat limited in composition. Here the authors use a computational strategy to predict the existence of a layered boride material, which they synthesize and demonstrate prospective for use as an anode material. Therefore, MAX phases possess the features of elastically stiff, strong, and heat-tolerant ceramics4, although their electrical and heat conductivities drop linearly with increasing temperature, as with a metal5. For the reported MAX phases, M represents an early transition metal, A is generally a metal element in group 13 or 14, while X is limited to C or N. Utilizing the significant difference in strength between the metallic M–A bonding and covalent M–X bonding, the A-layer can be selectively etched to form two-dimensional (2D) materials known as MXenes, which cannot be synthesized directly due to their thermodynamic metastability9. MAX phases and the derived MXenes have attracted extensive interest because of the abundance of their possible forms and structures, and their excellent chemical and mechanical stabilities, and thus, their broad applicability16. For instance, MXenes have shown great promise in applications involving electrochemical energy storage14 and catalysis16 because of their excellent conductivity, rich interlayer porosity and high surface functionalization. However, the question has been raised as to whether it is possible to synthesize new MAX phases and corresponding MXene materials without the limitation of using C and N as the X component.M18, 2D boron and related borides have attracted broad attention for their potential applications in nanoelectronic devices21. Therefore, the exploration of boron (B)-containing MAX phases and their derived MXenes with exotic properties is of considerable interest in this research field. Ade and Hillebrecht recently proposed several ternary borides as analogs of MAX phases by introducing B as the X element22. However, each boride presented in their work was orthorhombic, which is totally different from the hexagonal structures (P63/mmc symmetry) of known MAX phases. In MAX phases, the M, A, and X atoms alternately stack along a hexagonal close-packed (HCP) manner and respectively form equilateral triangles of their own  parallel to each other. However, M and A atoms in the reported borides alternately stack along orthorhombic manner. And the M atoms, which are coordinated with boron, form non-equilateral trigonal prisms, with the side edge along x direction determining the lattice constant a, perpendicular to the A layers. Furthermore, the nearest neighbor boron atoms in reported borides form one-dimensional zig-zag chains perpendicular to the A layers also. Therefore, these B-containing ternary compounds were not categorized as new MAX phases, but were instead named MAB phases. However, Ade and Hillebrecht did indicate the possibility of extending the family of MAX phases to B-containing compounds. However, it is difficult to explore ternary compounds using conventional techniques because the number of candidates is too large , even without considering the exponentially increasing number of reactions involved. Therefore, a feasible strategy to simplify the search for ternary compounds based on the available domain knowledge is in high demand.Recently, with the successful deposition of atomic boron layers on various metal surfaces21 have revealed that an abundance of stable or metastable 2D structures can be formed using Ti and B. Our preliminary study and ref. 21 both suggested that TiB and Ti3B4 can exist in the form of layered structures in which TiB (space group Cmcm) or Ti3B4 (space group Immm) layers are connected by Ti-Ti metallic bonds  is prohibited. Therefore, a possible strategy could begin from available layered TiB and Ti3B4 and involve inserting A elements into the interlayer Ti-Ti bonds to synthesize B-containing MAX phases, with subsequent etching of A to obtain the related MXenes. Although TiB and Ti3B4 are metastable compounds, the synthesis of a TixAyBz could be possible if the reaction enthalpy is negative with respect to TixBz , bulk A and other competitive phases.Recent investigations2InB2 and Ti2SnB2 as theoretically stable compounds at ambient pressure and a high pressure of 10 GPa, respectively. Based on theoretical predictions, Ti2InB2 was successfully synthesized through a solid-state reaction, and subsequently a layered TiB compound was obtained by the removal of indium through a high-temperature dealloying process. The obtained compounds were evaluated experimentally and were confirmed to be consistent with the predictions. The layered TiB was predicted to be a promising anode material for Li/Na ion batteries according to density functional theory (DFT) calculations. Pristine TiB monolayers possess a much higher ion (Li or Na) storage capacity than Ti3C2, a representative MXene, and exhibit a very low energy barrier for the diffusion of Li and Na ions.In the present work, we examine the possibility of forming boron-containing MAX phases by employing a computation-based strategy. The current strategy involves greatly improving the computation efficiency to identify TixAyBz compounds estimated through reactions of TixBz with A rather than using Ti, B, and A. In the present material design loop, this step was realized by conducting binary variable-composition searches as implemented in the USPEX code28 using TixBz and bulk A as ending compounds. A pre-investigation to identify the structure of TixBz was performed by employing a binary structure search in the Ti-B system , see details in Supplementary Table 2InB2 and Ti2SnB2 is higher than those of X/M ratios  in conventional MAX phases, boron atoms occupy the X sites between M layers and form a graphene-like layer  states between the d orbitals of Ti atoms and the p orbitals of B or In atoms are located below (above) the Fermi level, whereas the non-bonding states of Ti are located between these bonding and antibonding states (near the Fermi level), and predominantly contribute to the metallic nature of Ti2InB2. This electronic structure is qualitatively similar to that for conventional MAX phases. Electron localization function (ELF) calculations showed that electron accumulation occurs between adjacent boron atoms, which reveals the 2c–2e (two center-two electron) nature of the B–B bonds  and M (Ti) is much weaker than that between M (Ti) and X (B), and is similar to that for the conventional MAX phases that can be engineered to 2D MXenes. For a clear comparison, the separation energy for the Ti/Al(001) and Ti/C(001) interfaces of Ti2AlC was calculated to be 5.66 and 11.90 J m−2, respectively. The Ti–In bonding in the newly predicted MAX phases is much weaker than that for Ti-Al bonding in Ti2AlC. The Ti/In-to-Ti/B separation energy ratio is 39%, which is also much smaller than the Ti/Al-to-Ti/C ratio of 48%. Consequently, the present calculations reveal the possibility of obtaining 2-D MXenes from Ti2InB2 by selective removal of indium through an appropriate approach. Phonon dispersion calculations . The orthorhombic phase (Cmcm) that exhibits layered characteristics  and a small amount of TiB2 (6.3 wt%) remained, as shown in Fig. 2InB2 was crystallized in the predicted structure (space group Pm2). In a Rietveld analysis, the optimum fit was obtained by assuming a preferred orientation of (00l) for Ti2InB2, which suggested a material with a lamellar crystal structure. A laminar structure was clearly evident from scanning electron microscopy (SEM) observations, as shown in Fig. 2InB2 in the x–y plane, with hexagonal patterns recording the projection of In and Ti atoms on the plane, which is consistent with the prediction   data  that describes the contribution from conduction carriers, and β is the coefficient of the Debye T3 term (βT3) associated with propagating acoustic phonons.Tiion Fig. . A fast ata Fig.  were fit2InB2 powder in 50% HF for 12 h at room temperature. This resulted in total dissolution of the MAX phase, and even TiB2 was dissolved given sufficient time . The weight loss for the sample was in good agreement with the mass of In contained in the Ti2InB2 compound. Figure Cmcm), together with TiB2 as an impurity phase in the prepared Ti2InB2.Indium atoms were gradually extracted from Ti2 g−1 with increasing heating temperature, which also indicates that In species were gradually extracted from Ti2InB2  phase with a layered structure during removal of the In species. As shown in Supplementary Fig. Cmcm) phase to have originated from a phase change of hexagonal (P2) with a similar layered structure, which can be confirmed by the simulation results of FPMD at 1273 K for 20 ps . This means that the chance for the transition from Cmcm to Pnma can be little due to the weak thermodynamic driving force, which explains the experimental result that the metastable Cmcm phase is the dominant product by heat treatment at 1050 °C.The specific surface area increased from 2.45 to 6.16 mnB2 Fig. . Unfortuoms Fig. . An enlaoms Fig.  and the oms Fig.  inset ofoms Fig. . The , were present, a new layered material, TiB (Cmcm), was successfully prepared by dealloying the new Ti2InB2 MAX phase. Although the layered TiB (Cmcm) is not comparable with 2D MXenes, the successful removal of In atoms suggests the possibility to obtain TiB MXene at mild conditions. Therefore, it is necessary to investigate the potential applications of 2D TiB in advance. To evaluate the thermal stability of the 2D TiB structure at elevated temperatures for applications, we carried out FPMD simulations at 1273 and 1773 K for 10 ps  ↔ TiB + Li+(Na+) + e−. To confirm the potential of layered TiB as an anode material for Li/Na ion batteries, adsorption of Li and Na atoms on the surface of a TiB monolayer was first studied using DFT calculations. These calculations revealed that each TiB primitive cell (Ti2B2) can accommodate up to two Li or two Na atoms , which corresponds to a composition of TiBLi or TiBNa 11, Ti2C (359 mAh g−1 for Na+)36 and even higher than that of the commercial anode material, graphite (372 mAh g−1 for Li+)11. The open circuit voltage (OCV) for the intercalation reactions involving Li+ and Na+ ions on a TiB surface was respectively estimated to be as low as 0.33 and 0.17 V, respectively, which are both much smaller than that for Ti3C2 (0.62 V for Li+) and comparable with that for graphite (about 0.2 V for Li+) and the hypothetical TiC3 (0.18 V for Na+)36. The calculated ELF across the absorbed Li/Na atoms and TiB monolayer show that the electron cloud spread out in the metal layers can screen the repulsion between the positive metal ions , graphite (0.30 eV for Li diffusion), anatase TiO2 (0.35–0.65 eV for Li diffusion) and the predicted TiC3 (0.18 eV for Na diffusion)36. We noted that the diffusion activation energy for Li+ and Na+ ions on oxidized TiB surface gets increased (0.31~0.68 eV for Li+ and 0.22~0.50 eV for Na+) but is still competitive to the reported conventional materials  could be a promising alternative material to the commercial graphite anode in Li/Na ion batteries. Moreover, the diffusion of Li+/Na+ ions on hexagonal TiB surface was investigated  and a close-packed atom stacking sequence, the co-existence of weak Ti-In metallic and strong Ti-B covalent bonding along the [001] direction, metallic electrical resistivity and ceramic-like low heat capacity. With solid evidence from theoretical calculations and experimental validation, we have provided a clear example to extend the MAX phase family to boron-containing systems. Moreover, DFT calculations revealed that the separation energy for the Ti/In metallic interface is much smaller than that for the Ti/B covalent interface, which indicates the possibility of In atom removal while maintaining the TiB layered structure. Following this theoretical prediction, a layered TiB structure (Cmcm) was obtained by dealloying the Ti2InB2 MAX phase under vacuum conditions. The obtained TiB exhibited superior stability over the conventional MXenes and maintained its layered structure at high temperature. DFT calculations also predicted that the conductive TiB would be a promising anode material for Li/Na ion batteries with respect to a low OCV, a low Li/Na ion diffusion barrier, and a high theoretical Li/Na ion capacity. The present research will extend the fascinating class of MAX phases and MXenes.A progressive calculation-based approach was employed to predict and synthesize the boron-containing MAX phase Ti28 and the Vienna Ab initio simulation package (VASP)39. This approach was used to identify the structure with the lowest free energy for a given composition or in a composition range under given external conditions (0 K and 1 atm). Three types of structure searches were performed: fixed, binary variable composition, and ternary variable. For a fixed composition structure search, the calculation scheme comprised two steps: global optimization and local optimization. Structure initiation and variation were performed using USPEX in the global optimization step. For a variable composition structure search, two or three elements/compounds were adopted as reference components in a search of the target system. All possible combinations of the reference components were considered in the USPEX structure search, limited only by the number of atoms number per unit cell.Structure searches were conducted using a combination of Universal Structure Predictor: Evolutionary Xtallography (USPEX)40 k-point mesh solution in reciprocal space was 2π × 0.06 Å−1 for all structures. For structure refinement, the cutoff energy and k-point were, respectively, improved to 600 eV and 2π × 0.04 Å−1 to optimize the most stable structures obtained in the evolutionary structure searches. The atomic positions and unit cell volumes were fully relaxed until the total energy converged within 0.001 eV. These calculation settings were also adopted for the electronic structure calculations. Specifically, a denser k-mesh of 2π × 0.02 Å−1 was used for the density of states (DOS) for the predicted structures. Phonon dispersion calculations were performed to confirm the dynamic stability of all thermodynamically stable structures using VASP39 and Phonopy41. We performed first-principles molecular dynamics (FPMD) calculations using the VASP39 to simulate the transition from hexagonal TiB to orthorhombic phase and confirmed the thermal stability of 2D-TiB at elevated temperature. The FPMD simulations were performed with an NVT ensemble  with a Nosé thermostat. The time step of 2.0 fs used for each simulation. To compare their thermodynamic stabilities of different phases of TiB at different temperature, their Helmholtz free energies were calculated by following equation:In the evolutionary structure search, thousands of structures were relaxed using DFT calculations. Therefore, sufficiently good settings were used to reduce the calculation cost: the energy cutoff for the plane-wave basis set expansion was set at 400 eV, and the Monkhorst–PackEtot was calculated by VASP39, the entropy S(T) was obtained by VASP and Phonopy41 calculations.The total energy 2InB2 was synthesized from Ti (99.9%), In (99.99%), and B (99%) powder using a solid-state reaction route. The chemicals were purchased from Kojundo Chemical Laboratory Co., Ltd. Single-phase Ti2InB2 could not be directly obtained, because it competed with other stable phases (mainly TiB2 and Ti-In phases). Finally, the yield was maximized by varying many different experimental conditions: the optimized values were 1100 °C for 36 h without further annealing using a quartz tube sealed with Ar gas and Mo foil as the protector, and excess amounts of both Ti and In (>25%). The samples used for characterization and property measurements were prepared with increased amounts of Ti and In by 50%. More information regarding the optimization process of temperature, the type of crucible, the atmosphere, the annealing process and the initial composition ratio is provided in Supplementary Figs. −1, 100 mL) at room temperature for 10 h. Ti–In phases  were gradually dissolved with the evolution of H2 bubbles, and the color of the solution became purple (TiCl3). TiB2 exhibited high stability against etching, and could not be removed . Several SiC  ingots were placed separately. The molybdenum tube and SiC ingots were heated at 900–1050 °C (5 °C min−1) for 6 days in a dynamically evacuated quartz tube (about 10−4 Pa) as shown in Supplementary Fig. 2InB2, and the yield of TiB gradually increased with increasing annealing temperature. Finally, the optimized condition for the dealloying reaction was determined to be 1050 °C for 6 days. The obtained product TiB was a puce color, different from the dark gray color of the parent Ti2InB2.The as-prepared TiPowder X-ray diffraction (XRD) measurements were performed using an X-ray powder diffractometer with Cu Kα radiation . The morphology of the sample was evaluated using field-emission scanning electron microscopy  and the component elements were analyzed using energy-dispersive X-ray spectroscopy . Transmission electron microscopy (TEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images were obtained using a JEOL JEM-ARM200F atomic resolution analytical electron microscope operated at an accelerating voltage of 200 kV. Resistivity and heat capacity were measured with a physical properties measurement system . The Brunauer−Emmett−Teller (BET) specific surface areas of the samples were determined from nitrogen adsorption–desorption isotherms measured at −196 °C using an automatic gas-adsorption instrument .Supplementary InformationPeer Review File


Concentrations and δ15N of chlorophyll a, bacteriochlorophyll a, nitrate, and ammonium in benthic microbial mats indicate that inhibition of nitrification suppresses denitrification and anammox, resulting in efficient ammonium recycling within the mats and high primary productivity. We also suggest that the release of 15N-depleted NH3(gas) with increasing salinity enriches ammonium 15N in surface brine (≈34.0‰). Such elevated δ15N is also recorded in geoporphyrins isolated from sediments of the MSC peak (≈20‰), reflecting ammonium supply sufficient for sustaining phototrophic primary production. We propose that efficient nutrient supply combined with frequent bottom-water anoxia and capping of organic-rich sediments by evaporites of the Mediterranean MSC could have contributed to atmospheric CO2 reduction during the late Miocene.The biogeochemistry of hypersaline environments is strongly influenced by changes in biological processes and physicochemical parameters. Although massive evaporation events have occurred repeatedly throughout Earth history, their biogeochemical cycles and global impact remain poorly understood. Here, we provide the first nitrogen isotopic data for nutrients and chloropigments from modern shallow hypersaline environments  and apply the obtained insights to δ The biogeochemical cycle of hypersaline environments is highly distinct from that of freshwater and marine conditions because it is strongly affected by changes in the physicochemical parameters of the brine as well as in the state and composition of the biological community in response to salinity variations. In spite of the energy cost to overcome the salt stresses imposed on cellular systems4, hypersaline environments can be highly productive5. This is especially the case for shallow hypersaline environments, where densely populated microbial mats inhabited by a range of physiological groups of microorganisms are formed . Constraining the biogeochemical cycle, which is responsible for the diversity of the niche space6 and the primary productivity7, is essential for understanding such ecosystems. Moreover, considering that hypersaline environments can become surprisingly productive, the massive evaporation events that occurred repeatedly throughout Earth’s history8 could have had a global impact. However, the biogeochemical cycles of the massive evaporation events remain poorly constrained.Present-day and past hypersaline environments are unique and extremely important settings that affect biology, climate, and global geochemistry2-fixation, nitrification, denitrification, anaerobic ammonium oxidation [anammox]). It strongly influences photosynthetic primary productivity, and thus the entire biological community, by modifying the amount and chemical species of the bioavailable nitrogenous nutrients9. Because hypersaline microbial mats are often highly productive5, the nitrogen cycle in these systems is expected to operate such that the supply of bioavailable nitrogen is sufficient to maintain active photosynthesis. Various approaches have been applied to study nitrogen cycle, such as in situ measurements, culture and incubation experiments, and genetic analyses; some studies report the importance of N2-fixation11, whereas others have demonstrated that ammonium12 and dissolved organic nitrogen13 derived from the degradation of organic matter are crucial as nitrogen sources.Nitrogen has an active redox cycle comprised mainly of biological processes  of nitrogenous nutrients and organic compounds enable us to elucidate and quantify the relative importance of the processes controlling the flow of nitrogen15. Significantly, the isotopic signatures in modern hypersaline environments can be used for interpreting the δ15N signatures of the analogous ancient environments. In particular, the isotopic compositions of chloropigments (chlorophylls and bacteriochlorophylls) record primary signals of the physiology of phototrophs and the nitrogen substrates that they assimilate18. Moreover, the preservation of chloropigments as geoporphyrins in sediments on a geological timescale make them an ideal tool for reconstructing past nitrogen cycles22.The nitrogen isotopic compositions  and gypsum (CaSO4·2H2O) ponds, where benthic microbial mats are present, and halite (NaCl) ponds, which lack a benthic ecosystem , Culcasi (CU), and Chiusicella (CH) salterns Fig. . Three stem Fig. . The degws Table :1\documeMg cancelled out the effect of the condensation of solutes during evaporation and thereby allowed us to examine the addition or removal of the solutes  than for bacteriochlorophyll a (−8.1‰ to 0.1‰) in all ponds ) also showed similar depth profiles in the studied ponds. Ammonium was the largest pool of dissolved inorganic nitrogen (334.3–558.7 µmol L−1); its concentrations were more than an order of magnitude higher than those of the surface-brine nitrate, ammonium, and porewater nitrate  ranging from −4‰ to −20‰ in laboratory culture experiments under freshwater to marine conditions34. Our rough estimates of ε for phototrophic assimilation of ammonium inside microbial mats show ε values for purple sulfur bacteria (εPSB) of −10‰ to −5‰ for CU-6, −15‰ to −10‰ for CU-7, and −18‰ to −12‰ for CU-8  to the halite pond (CU-5) was around −22‰, with ε in the carbonate and gypsum ponds ranging from −5‰ to −20‰  to the atmosphere due to the decrease in gas solubility along the salinity gradient. A laboratory study has suggested that the release of NH3(gas) from fluids occurs episodically; between these release episodes the gas stays in the fluids and maintains isotopic equilibrium, with NH3(aq) depleted in 15N relative to NH4+ by 45.4‰ at 23 °C38. Although the pH decline from 8.5 in carbonate ponds to 7.0 in halite ponds  from around 10% to less than 1%, continuous evaporation, inducing repeated degassing and subsequent dissociation under isotopic equilibrium, could result in a progressive δ15N increase in the remaining ammonium. We expect that 15N-enriched ammonium can be used as an indicator to constrain the nitrogen cycle of hypersaline environments, where ammonium is abundant and partially dissociates to NH3(aq).We suggest that the δnds Fig.  is assoc40). Its peak occurred during a short time window  and was accompanied with the precipitation of huge volumes of primary halite in the deep Mediterranean settings. Here, we measured the δ15N of geoporphyrins purified from the mud–anhydrite layers between the halite layers of the Realmonte salt mine , which were deposited in the Caltanissetta basin during the MSC peak , the Mediterranean Sea experienced a massive evaporation event referred to as the Messinian salinity crisis  during the arid season  would reveal whether efficient carbon sequestration in marginal basins is a local process or widespread in the Mediterranean, thereby clarifying the impact of the MSC on the global carbon cycle.The role of the Mediterranean MSC as a carbon sink needs to be explored because it has important implications for the remarkable shifts in the global carbon cycle during the late Miocene. This period is characterized by a decline in the atmospheric CO15N signatures of chloropigments in the benthic microbial mats is expected to be important comparison data for elucidating the nitrogen cycle of ancient analogous environments such as the stromatolites, which strongly influenced Earth’s surface biogeochemistry3. Moreover, the substantially high δ15N of ammonium formed during evaporation is useful in clarifying the nitrogen cycle of hypersaline environments: we interpret the extremely high δ15N of geoporphyrins from the MSC peak in the Caltanissetta basin as reflecting a supply of ammonium from the subsurface anoxic brine sufficient to sustain phototrophic primary production. In combination with the frequent formation of bottom water anoxia and capping of organic-rich sediments by evaporites, our first insights into the nitrogen cycle during the MSC imply efficient carbon sequestration, which might have contributed to the decrease in atmospheric CO2 during the late Miocene.Our study highlights the dynamic variations of the nitrogen isotopic signatures in solar salterns, suggesting that the high primary productivity of the hypersaline microbial mats is supported by efficient recycling of ammonium resulting from the suppression of nitrification, denitrification, and anammox. This first report of the highly distinct δThree commercial solar salterns at Trapani  were investigated: the Sosalt (SS), Culcasi (CU), and Chiusicella (CH) salterns Fig. . These s3) ponds, gypsum (CaSO4·2H2O) ponds, and halite (NaCl) ponds. The carbonate ponds , in which calcium carbonate precipitates, are characterized by the formation of a dense benthic microbial mat . The purged N2O was then introduced into a gas chromatograph  equipped with a PoraPLOT column  and a GC III Interface (Thermo Finnigan) to purify the N2O. The nitrogen isotopic composition of the purified N2O was measured with an isotope ratio mass spectrometer . The isotopic data was calibrated with three isotopic nitrate standards . The isotopic compositions are expressed in conventional δ notation relative to AIR. The analytical precision was within 2.0‰ (±1σ).Our experimental procedures largely followed those reported previously4+ and NH3(aq)) as (NH4)2SO42− 56. The (NH4)2SO42− was then converted to NO3− with the persulfate oxidation method57. The nitrogen isotopic composition of the NO3− produced was measured with the microbial denitrifier method. The measured δ15N of N2O was calibrated to that of total ammonium using three ammonium isotopic standards  that were analyzed concurrently with the samples. The analytical precision was within 1.0‰ (±1σ). The concentration of total ammonium was measured with a fluorometric or colorimetric technique; i.e., the o-phthalaldehyde method58 for the surface brine of CU-6, -7, and -8, and the indophenol blue method59 for all other samples.We used the diffusion method to recover the total ammonium  connected to a Thermo Finnigan Delta plus XP IRMS via a ConFlo III Interface60. Based on repeated measurements of our laboratory standards, the analytical error was estimated to be within 0.6‰ (±1σ).For the bulk δ23. In brief, the surface deposits of the microbial mats , and the yellowish transparent, green, and pink layers of the gypsum crusts (SS-1 and CH-1) were each freeze-dried and ground to powder. The samples were extracted three times with acetone by sonication, and then extracted with n-hexane. The n-hexane fraction was dried completely under N2 gas and dissolved in 100 µL of N,N-dimethylformamide for HPLC analysis. All procedures were performed in a dark room.The analytical procedure used for the purification of chloropigments followed that described previously−1 and the column temperature to 30 °C. The collected chlorophyll a and bacteriochlorophyll a were dissolved in 1.5 mL of hexane and reacted with 2 M HCl to convert them to pheophytin a and bacteriopheophytin a, respectively for the second purification step with HPLC. The column used for the second purification step was an Agilent Zorbax Eclipse PAH column . The pigments were eluted isocratically with 80% acetonitrile: pyridine  and 20% ethyl acetate: pyridine  for 5 min, followed by a linear gradient of ethyl acetate: pyridine to 60% over 25 min, and a linear gradient of ethyl acetate: pyridine to 100% over 10 min. The flow rate was set to 1 mL min−1 and the column temperature to 15 °C.The pigments were isolated and purified with dual-step HPLC using an Agilent Zorbax Eclipse XDB-C18 column . The pigments were eluted isocratically with 75% acetonitrile: pyridine  and 25% ethyl acetate: pyridine  for 5 min, followed by a linear gradient of ethyl acetate: pyridine to 50% over 50 min. The flow rate was set to 1 mL min59. The purified chloropigments were dissolved in dichloromethane, transferred to precleaned smooth-wall tin capsules, and dried before analysis. Based on repeated measurements of our laboratory standards, the analytical error was estimated to be within 0.7‰ (±1σ).The stable nitrogen isotopic compositions of the chloropigments were measured using a modified EA/IRMS62. The salt unit has been divided into four main lithological units from base to top: A–D64. Unit C, investigated in this study, was deposited after the basin had undergone desiccation64 and is composed of more than 120 lithological cycles, each one represented by the superposition of three facies: (i) a millimeter-thick mud layer; (ii) a millimeter–centimeter-thick anhydrite (CaSO4) or polyhalite (K2MgCa2(SO4)4·2H2O) layer; and (iii) a decimeter-thick halite layer with white patchy polyhalite grains  and “Rosone section” (towards the bottom of Unit C)41, where Unit C mud–anhydrite–halite triplets are observed. Before the analysis, the halite layer of the rock sample was largely removed with a chisel and hammer. The remaining mud–anhydrite layer of the rock sample was washed in Milli-Q water, methanol, and dichloromethane for 1 min each with sonication to eliminate any possible contamination on the surface of the rock.Fallen rocks consisting of a mud–anhydrite layer sandwiched between halite layers Fig.  were col66. The geoporphyrins contained in the pulverized sediment samples were extracted with dichloromethane: methanol . The extract was then separated with silica gel column chromatography and the low-polarity fraction was collected with dichloromethane. This was further separated into six subfractions with another round of silica gel column chromatography; the reddish-colored band was collected with dichloromethane: methanol .The analytical procedure for the analysis of geoporphyrins was modified from a previously reported procedureN,N-dimethylformamide: pyridine: acetic acid  at a flow rate of 1 mL min−1 and a column temperature of 20 °C. For the second purification step, a normal-phase HPLC analysis was conducted with an Agilent Zorbax SIL column . The mobile phase for the isocratic analysis was n-hexane: dichloromethane: N,N-dimethylformamide: pyridine: acetic acid  at a flow rate of 1 mL min−1 and a column temperature of 40 °CThe individual porphyrins were isolated and purified with dual-step HPLC. A reverse-phase HPLC analysis was performed with an Agilent Zorbax SB-C18 column . The porphyrins were eluted isocratically with acetonitrile: The measurement of the stable nitrogen isotopic compositions of the porphyrins was similar to the measurement of the chloropigments. To obtain enough samples for isotopic measurements, the purified peaks were combined for each sample. Based on repeated measurements of our laboratory standards, the analytical error was estimated to be within 0.5‰ (±1σ).3  for 2 h under 130 °C to release the chelated metal. The relative standard deviations based on the replicate measurements of the sample were within 2.5% for the monitored elements (±2σ).The metal chelated in the porphyrins was determined with a quadrupole ICP-MS  using the metals standard solution VII . The purified porphyrins were combined and reacted with 3 M HNOSupplementary materials


Subjective cognitive decline (SCD) patients are considered as a risk population for preclinical Alzheimer's Disease (AD). Supporting this idea, previous studies in SCD populations report subtle alterations in various cognitive and neuroimaging biomarkers that are typically affected during AD progression. To extend these observations, the present study examined whether SCD patients show atrophy of cholinergic basal forebrain nuclei (chBFN), analogous with recent findings in prodromal and clinical AD patients. We assessed volume reductions of the chBFN in 24 SCD subjects compared to 49 matched controls on 3D-T1-weighted MR images based on a postmortem derived atlas. Furthermore, we assessed whether chBFN atrophy was linked with cognitive, structural and metabolic biomarker alterations we previously reported in this SCD cohort: Using correlation analyses we tested for associations between the volumes of the chBFN with the hippocampal gray matter volume, and posterior medial glucose consumption, and the trajectory of verbal memory performance. The SCD cases showed a significant total volume reduction of the chBFN, with largest effect sizes in the Ch1/2 and Ch4p subdivisions of the chBFN. The latter was associated with a reduced glucose metabolism in the precuneus for the SCD group only. These data show an early involvement of the cholinergic basal forebrain nuclei in SCD predominantly in Ch1/2 and Ch4p which supports the conceptual link between SCD and preclinical AD. •Cholinergic forebrain shows atrophy in SCD.•Ch12 and posterior part of Nucl. Basalis Meynert (Ch4p) show largest effect size.•Atrophy of the Ch4p correlates with reduced glucose metabolism in precuneus. FrequenIt is well established that AD is associated with cell loss of the cholinergic basal forebrain nuclei leading to a reduced cholinergic innervation of the cortex . The choMRI-based volumetry has shown that gray matter volume of the chBFN declines from prodromal to clinical stages of AD , which iCritically, a correlation of basal forebrain volumes with amyloid deposition in cognitively normal and mildly impaired non-demented elderly suggests that chBFN alterations may already be present in preclinical at-risk individuals with confirmed AD pathology . MoreoveIn this study, we measured chBFN volumes in a previously reported sample of SCD subjects who predominantly report subjective memory impairment . Based oWe expected that this atrophy would be associated with metabolic and structural markers of AD-typical neurodegeneration, as well as with declining memory performance over time.22.1n = 24) were recruited by the memory clinic of the Clinical Treatment and Research Center for Neurodegenerative Disorders (KBFZ), Departments of Psychiatry and Neurology, University Hospital Bonn. The definition of SCD was based on the fact that subjects were referred to the memory clinic for work-up of memory complaints and on a standard question: ‘Do you feel like your memory is becoming worse?’ Possible answers to this question were: ‘no’, ‘yes, but this does not worry me’ and ‘yes, this worries me’. To be classified as SCD in this study, the subjects had to answer with ‘yes, this worries me’. We only included subjects whose memory worsening was confirmed by others  was recruited from the general population. Participants were included as controls if they denied any memory worsening or if a memory decline was only reported on active inquiry and was considered of no concern.The control cohort  neuropsychological battery . For bot2.2The study protocol was approved by the ethics committee of the medical faculty of the University of Bonn. It was performed in accordance with the Declaration of Helsinki. All participants provided written informed consent.2.3The neuropsychological test battery was designed to test verbal and visual memory, processing speed and executive functions, and was administered at baseline and at follow-up visits.Verbal memory was assessed with the Verbal Learning and Memory Test (VLMT) , the GerBoth groups returned to the memory clinic for three follow-up visits including neuropsychological testing (see below). On average, the SCD group returned 14 ± 3.0 months, 32.5 ± 6.1 months, and 45.1 ± 5.6 months after baseline, while the control group returned after 15.5 ± 2.9 months, 33.3 ± 6.1 months, and 41.7 months. Twenty-two SCD subjects had at least one follow-up, 16 had two follow-up visits and 11 had three follow-up visits. Forty-five participants of the control group returned for at least one follow-up visit, 36 participants had two follow-up visits, and 15 had three follow-up visits.2.42, matrix size 320 × 320, number of slices = 170, spatial resolution 0.8 × 0.8 × 0.8mm3).Structural data were obtained from 24 SCD subjects and 49 controls. All scans were performed on a 3 Tesla MR system , equipped with an 8-channel SENSE head coil. For each participant, four high-resolution T1-weighted data sets were acquired consecutively, using a 3D turbo field echo (TFE) sequence . Preprocessing included realignment and averaging of each participant's four structural data sets, tissue class segmentation, and spatial normalization to MNI standard space using the high-dimensional registration algorithm DARTEL  of gray matter volume for the right hippocampus was performed following a protocol described previously  calculat2.4.2The cholinergic basal forebrain is composed of four different cell groups, which form partially overlapping ‘subunits’ (Ch1–Ch4), and project to different anatomical brain structures . Ch1 reftot) was calculated by adding up the volumes of all subdivisions. All volumes were normalized by the ICV as described above.Due to the small size, Ch1 and Ch2 were not considered separately and will be referred to as ‘Ch1/2’ in the following. We also combined Ch4am, Ch4al without NSP, and Ch4i into one volume (Ch4). The posterior region (Ch4p) and NSP were considered separately, and the ROIs were combined across both hemispheres for all of the nuclei. As described for the hippocampal volume, the individual chBFN volumes were calculated by summing the voxel intensity values of the GM partition across the chBFN ROIs. The total volume of the basal forebrain nuclei (chBFN2.518F] fluoro-2-deoxyglucose PET (FDG-PET) scans were performed on a PET/CT scanner . Imaging started 30 min after injection of 150 MBq FDG. The acquisition started with low dose CT-scan for attenuation correction . PET emission data acquisition was performed over 20 min. The subsequent attenuation correction for PET was based on a rescaling of the CT image and scatter correction using a single scatter model. Emission data were reconstructed with an in-plane spatial resolution of 1.3 × 1.3 mm2 and a slice thickness of 2.4 mm. The intrinsic resolution of the Biograph system is 9.3 mm  we used a one-sided independent two sample t-test with the a priori hypothesis of smaller volumes in the SCD group. In a second step, we tested which subnucleus was affected and determined the respective effect sizes.To test whether the SCD group showed a decreased total volume of the basal forebrain nuclei . To exp2.6.5In order to investigate the association between chBFN volume reduction and our second putative marker of AD-related degeneration, we performed a correlation analysis in the SCD group between the significantly atrophied basal forebrain nuclei and the right hippocampus.For any correlation yielding a significant effect, we conducted complementary analyses for the control group in order to test the specificity of the finding. Group differences were tested using linear regression analysis.2.6.6To examine potential associations between chBFN volumes and regional glucose metabolism, similar analytic procedures as for the structural MRI data were applied. The right precuneus served as target region.33.12(3) = 2.67, not significant). No significant difference with regard to age, gender, and years of education existed between those participants still remaining in the study and those dropping out.All participants scored within 1.5 standard deviations (SD) of the mean according to age-, gender-, and education adjusted normstot or its subnuclei were not statistically significant.The SCD group scored slightly lower in the MMSE but was still well in the range of normal cognition  compared with the  volumes of the control group (mean ± SD = 642 ± 102 mm3). This difference of 42 mm3 was significant , with a small to moderate effect size (Cohen's d = 0.45).All chBFN volumes were normally distributed for both groups and homogeneity of variances was met according to Shapiro-Wilk and Levene's Test, respectively. The  chBFN3 vs. 91 ± 19 mm3; t71 = 2.29, p = 0.013, d = 0.57 for Ch1/2, and 99 ± 15 mm3 vs. 106 ± 15 mm3, t71 = 2.01, p = 0.024, d = 0.50, for Ch4p, respectively).Following up the significant overall effect, the Ch1/2 and Ch4p nuclei showed significantly smaller volumes in the SCD group as compared with the control group, with moderate effect sizes . The main results are summarized in The analyses for the other nuclei yielded no significant effect, although a smaller group difference in Ch3 volume approached significance (t3.3SCD = −0.033 ± 0.017), compared to a slight improvement for the control group (slopeControl = 0.150 ± 0.123). The group difference was significant . Neither for the control group nor for the SCD cohort we found any significant correlations between chBFN volumes and the memory performance over time.Modeling memory performance over time using a growth curve model revealed a significant memory decline over time for the SCD group .No significant correlation was found between Ch1/2 and right hippocampal volume . For bot3.5Ch1/2 did not show any significant correlations with metabolism in the respective ROI for any single group, or after pooling the data across both groups.group = 3.739, p = 0.0004; see also p =0.053). The difference of the correlation coefficient was significant .Meanwhile, Ch4p showed a significant association with right precuneus metabolism for the SCD group only  and yiel4In the present study, we used in vivo volume measurements of cholinergic forebrain nuclei in SCD and correlated the findings with memory trajectory over time, hippocampal volume and glucose metabolism of the precuneus. Consistent with the idea that SCD patients show a higher risk of preclinical, AD-related neuroimaging and cognitive alterations, we were able to show that the cholinergic basal forebrain volume was smaller in SCD compared to controls matched for age, gender and ApoE status, consistent with previous findings in MCI and AD dementia. Even though almost every nucleus showed atrophy, greater effect sizes were observed for Ch1/2 and Ch4p than for Ch3. While these atrophic changes were not predictive for the cognitive trajectory of the SCD patients, they were related to other AD-related neuroimaging markers that we previously found to be altered in this cohort . We showThe observed basal forebrain atrophy is in line with postmortem and in vivo studies in prodromal and clinical AD patients: Neuropathological data show that AD is associated with a loss of cholinergic neurons of the chBFN , and a cA relationship between the Ch4p alterations in our SCD patients and AD-related neurodegenerative mechanisms is further supported by the observed correlation between Ch4p volume, and the glucose metabolism the right precuneus, which was only observed in the SCD group. The lower the volume of the Ch4p the lower the glucose consumption in the right precuneus. The exploratory data analysis using a bilateral precuneus ROI revealed a similar result. Even though the effect seems to be driven by the right precuneus a larger group size might be needed to further elucidate any laterality effect. However, the correlation between the Ch4p volume and the right precuneus metabolism is a remarkable finding, because lower precuneus glucose metabolism is known to be a highly sensitive and early marker for the development of AD . A detecThe link may be indirect, i.e. depend on the mediation (or common causation) of unidentified additional brain mechanisms. Here, integration with connectivity-based methods like resting-state fMRI e.g.  may helpWhile the observed associations of basal forebrain volumes with our second putative neuroimaging marker for AD neurodegeneration, right hippocampal GM volume, seem to provide further support for this claim at first sight, caution is warranted, since significant correlations were observed in both SCD and control participants: Accordingly, they may reflect a common non-AD process, e.g. aging.Despite the agreement of the presented Ch4p/NBM atrophy in SCD with findings reported in MCI and AD, and the relation with local glucose metabolism, our data also show distinct differences regarding the role of Ch1/2 with respect to the literature. While reports on MCI and early-stage clinical AD published so far  support 4.1The reported data in this study are based on in vivo MRI measurements of structures that do not have a sufficient MR contrast to its neighborhood, and thus the derived chBFN volumes rely on a recently developed cytoarchitectonic atlas that was based on postmortem data of a single subject . Given tOne might argue that groups differences in basal forebrain volumes were actually driven by confounding group differences in depression level, which represent an independent, non-neurodegenerative influence on SCD symptomatology. In general, all participants' BDI scores were within the normal range, and none of the participants reached clinical levels. Moreover, we did not find any significant correlation between the BDI scores and any cognitive measures or imaging marker see: , includiThe sample size can be considered as an additional limitation. Even though the group sizes were sufficiently high to detect statistically significant differences in chBFN volumes, a larger group size might have allowed to elucidate further the role of the Ch1/2 atrophy within the SCD group. As an additional point one might argue that the tests used to compare the volume differences between both groups were one-sided and the reported data are not corrected for multiple comparisons. However, considering histology data and imaging data at preclinical stages, testing for atrophy only can be justified. In addition, the main findings of this paper, the atrophy of the cholinergic basal forebrain and significant correlation between Ch4p volume and precuneus hypometabolism in SCD, would still hold if one would only test for changes of the BF-total volume - ignoring the substructure of the cholinergic system- and perform the correlation analysis with those subregions showing the nominally largest volume reduction .Also, the irregular numbers of follow-up visits and intervals in-between are not optimal. However, by using a growth curve model to determine the individual memory trajectory, we addressed this problem adequately. Unfortunately, the observation time was still too short to clearly associate the selected cognitive and neuroimaging parameters with individual conversion to dementia which would prove the underlying assumption that they represent actual biomarkers for preclinical AD progression. For this, the required observational time periods are estimated to be in the range of 10–15 years in samples that start at the SCD stage .Of course, confirmation of preclinical AD in SCD patients does not require clinical long-term follow-up but could be achieved by in vivo measurements of AD pathology, i.e. CSF or PET examinations of amyloid burden. Unfortunately, this sort of information is not available for the present cohort. Yet, our initial findings may provide a starting point for further testing in larger-scale longitudinal study cohorts which also provide additional amyloid measures, e.g. the Harvard Aging Brain Study .5To the best of our knowledge, the presented data show for the first time that chBFN atrophy is already present in SCD which would support the idea of SCD as an at-risk population for preclinical AD. This notion is further supported by an association between Ch4p atrophy and decreased glucose metabolism in the right precuneus in the SCD group. These findings may provide a good starting point for larger-scale studies which also take into account direct measures of AD pathology, and longer follow-up durations to monitor the long-term clinical outcomes of this risk population.


The aim of the study was to investigate whether intravenous (iv) infusion of exenatide, a synthetic GLP-1 receptor agonist, could provide a protective effect against myocardial ischemia-reperfusion injury after coronary artery bypass graft (CABG) surgery.−1 followed by a constant infusion of 0.025 µg min−1) (exenatide group) or iv insulin therapy (control group) for blood glucose control (target range 100–139 mg dl−1) during the first 48 h after surgical incision. All serum levels of troponin I measured during routine care in the Cardiac Surgery ICU were recorded. The primary outcome was the highest value of plasma concentration of troponin I measured between 12 and 24 h after ICU admission. The proportion of patients presenting an echocardiographic left ventricular ejection fraction (LVEF) > 50% at the follow-up consultation was compared between the two groups.A sub study analysis of patients > 18 years admitted for elective CABG and included in the ExSTRESS trial was conducted. Patients were randomized to receive either iv exenatide  [95% confidence interval (95% CI)] 0.16 , p = 0.54). The highest troponin value measured during the first 72 h in the ICU was 6.34 [1.36–10.90] versus 5.04 [2.39–7.18] µg l−1, in the control and exenatide groups respectively . At the follow-up consultation, 5 (12%) versus 8 (16%) patients presented a LVEF < 50% in the control and in the exenatide groups respectively .Finally, 43 and 49 patients were analyzed in the control and exenatide groups, respectively {age: 69 [61–76] versus 71 [63–75] years; baseline LVEF < 50%: 6 (14%) versus 16 (32%) patients; on-pump surgery: 29 (67%) versus 33 (67%) patients}. The primary outcome did not significantly differ between the two groups (3.34 [1.06–6.19] µg lPostoperative iv exenatide did not provide any additional cardioprotective effect compared to iv insulin in low-risk patients undergoing scheduled CABG surgery.Trial registration ClinicalTrials.gov Identifier NCT01969149, date of registration: January 7th, 2015; EudraCT No. 2009-009254-25 A, date of registration: January 6th, 2009 Temporary interruption of coronary artery blood flow during coronary artery bypass graft (CABG) surgery can cause myocardial lesions during both the ischemic and the reperfusion phases. Brief episodes of ischemia before (pre-conditioning) or after (post-conditioning) interruption of the coronary blood flow is believed to activate protective intracellular mechanisms, thus preventing the induction of apoptosis and subsequent death of cardiomyocytes . SeveralExenatide is a synthetic GLP-1 receptor agonist. Intravenous (iv) administration of exenatide during the first 6 h after myocardial infarction has been shown to reduce infarct size –17, throThe hypothesis of this study was that iv exenatide could provide pharmacologic postconditioning against myocardial ischemia-reperfusion injury, and thus improve left ventricular function and quality of life after CABG surgery. The aim of this study was to investigate whether iv perioperative administration of exenatide would reduce cardiac enzymes release resulting from ischemia/reperfusion injury in patients undergoing scheduled CABG surgery.This is a sub study of the single-center, randomized, open-label, phase III/IV ExSTRESS trial, performed from January to December 2015. The aim of the ExSTRESS trial was to investigate whether perioperative iv administration of exenatide improved glycemic control in patients undergoing scheduled CABG surgery, and the main findings have previously been reported elsewhere /2, with Xn, the troponin I value measured at time Tn, and Xn−1, the last troponin I value measured at time Tn−1. We calculated the proportion of patients with BNP0–12 ≥ 200 pg ml−1, BNP12–24 ≥ 200 pg ml−1 or BNP24–48 ≥ 200 pg ml−1.Troponin I values were classed into 4 time intervals, based on admission to the Cardiac Surgery ICU, namely: 0–12 h  was measured by transthoracic echocardiography at the post-operative follow-up between 3 and 6 months after surgery. The proportion of patients with LVEF < 30%, 30% < LVEF < 50%, and LVEF > 50% at post-operative follow-up was recorded; the categories used are based on those used to calculate the EuroScore value. The administration of catecholamines  during the stay in the Cardiac Surgery ICU was also noted.To assess quality of life, the Short Form (SF) 36 questionnaire was completed by patients at the inclusion visit, and then sent to patients by post for completion at 1, 3, 6 and 12 months after surgery. The SF36 questionnaire is validated in the French language  and is r12–24). Troponin12–24 was considered to be the serum level of troponin at postoperative day 1 depending on whether the surgery was performed on the morning or in the afternoon. Serum level troponin at postoperative day 1 was associated with midterm and long-term outcome after CABG in previous studies   or medians (interquartile ranges [IQR] 25–75%)  and qualitative variables as number (percentage) unless otherwise stated. Standardized differences were used to assess imbalances between baseline characteristics. Comparisons between the control and exenatide groups were performed using the Chi square or Fisher’s exact test for qualitative variables, and the Student All analyses were performed using SAS version 9.4 . All statistical tests were 2-sided and a p-value < 0.05 was considered statistically significant.The present study is a sub study of the ExSTRESS trial. The ExSTRESS study was a randomized phase II/III study. The phase II methodology was performed according to a 2-stage procedure as described by O’Brien and Fleming and has been published elsewhere , 28. TheThe original intent of the present study was to assess whether iv exenatide could improve the quality of life after CABG by allowing a better left ventricular function while reducing myocardial ischemia-reperfusion injuries. A sufficient statistical power to test this hypothesis was anticipated by including all patients included in the phase II and in the phase III of the ExSTRESS trial. However, the ExSTRESS study was prematurely terminated for futility during phase II, in the absence of any clinically relevant benefit yielded by the use of exenatide to control blood glucose in patients undergoing CABG surgery. The sample size included in the phase II of the ExSTRESS study did not provide enough statistical power to compare quality of life between the control and exenatide groups. Thus, the primary hypothesis of the present study was adjusted to investigate the postconditioning effect of iv exenatide after CABG.The analyses in the current study were performed on the ExSTRESS study population but excluding patients who died in the Cardiac Surgery ICU, patients who had fewer than 3 troponin measures, and patients who received neither insulin nor exenatide. No sample size calculation was performed before data analysis.In the ExSTRESS study, a total of 297 patients were eligible during the study period. Fifty-five patients were randomized to each group, and 51 and 53 patients were analyzed in the control and exenatide groups respectively. For the present analysis, 8 and 4 patients were excluded from the control and exenatide groups respectively. The reasons for exclusions are detailed in the study flowchart shown in Fig. 12–24 did not significantly differ between the control (3.34 [1.06–6.19] µg l−1) and the exenatide (2.64 [1.29–3.85] µg l−1) groups   and plasma concentration of troponin I during the first 72 h in the Cardiac Surgery ICU were not significant different between groups . The median number of troponin assays performed during the ICU stay was 5 [4–6] versus 5  in the control versus exenatide groups (p = 0.55).Plasma concentration of troponin0–12, BNP12–24 or BNP24–48 ≥ 200 pg ml−1 are given in Fig. Plasma concentrations of BNP during the first 48 h in the ICU after surgery did not significantly differ between the control and exenatide groups  in the exenatide group received dobutamine during the ICU stay .The number of patients with LVEF < 50% at the follow-up consultation was 5 (12%) versus 8 (16%)  in the control and exenatide groups, respectively. No patient had LVEF < 30% at follow-up.The values of the dimensions of the SF36 questionnaire at inclusion, 1, 3, 6 and 12 months were not significantly different between the control and exenatide groups (data no shown).This sub study of a randomized clinical phase II/III trial suggests that iv exenatide for perioperative glycemic control after scheduled CABG surgery does not procure any cardioprotective effect and does not appear to improve cardiac function in the short or medium term.RISK) pathway [+-ATP channels [The cardioprotective effect of GLP-1 receptor agonists or GLP-1 metabolites has been largely explored and reported in animal models of ischemia-reperfusion injury –10, 29.  pathway , 6, 19, channels . In a rachannels , 16 repo−1, i.e. within an average time of 3 h after admission to the Cardiac Surgery ICU [Firstly, the modalities of intravenous administration of exenatide in our study were fixed by monitoring of blood glucose, with treatment initiated as soon as the first blood glucose value exceeded ≥ 140 mg dlgery ICU . Most stgery ICU , 16, exegery ICU , 16. In gery ICU . In contgery ICU  failed tBeyond the delay from the start of reperfusion, the beneficial effects of GLP-1 administration during reperfusion could be independent of the GLP-1 receptor pathway, and involve the GLP-19–36) amide, which results from the breakdown of GLP-1 by the plasma enzyme dipeptidyl peptidase-IV 6 amide, . This hySecondly, the dose of exenatide prescribed in our study could be insufficient to generate a significant cardioprotective effect. The dosing regimen used in this study was mandated by the national agency for the safety of health products , because the use of intravenous exenatide in our study was off-label. Therefore, the dose regimen imposed was that used in the only study of exenatide for the control of stress hyperglycemia available at the time of the design of our study . While iThirdly, the body of literature reporting protective effects with GLP-1 analogues comprised mostly studies comparing the study drug to a control group treated by placebo. In our study, all patients in the control group received insulin. A reduction in ischemia-reperfusion injury has been described with the use of insulin –37, whicInotropic effects of GLP-1 that could improve quality of life and functional symptoms has been reported in patients with chronic heart failure , in patiThis study suffers from some limitations that deserve to be underlined. Firstly, this is a sub study of a phase II/III randomized trial, and thus, the mode of administration of exenatide was not originally designed for the purposes of evaluating the potential cardioprotective effect of exenatide. Furthermore, the inclusion criteria of the main study selected a population of patients at low risk of post-operative heart failure. We therefore cannot rule out the possibility that different results might be observed if the dose regimen of exenatide or if the patient profiles were different. Secondly, no proper sample size calculation could be performed prior to the analysis in the absence of data in cardiac surgery patients, and the study suffered from a lack of power. Based on our results, 800 patients in each group would be required to reach a statistically significant difference in the primary outcome.This sub study of a randomized, phase II/III trial failed to observe any additional cardioprotective effect of iv exenatide compared to iv insulin following CABG surgery in a population of patients at low risk of post-operative heart failure. Both iv insulin and iv exenatide might have activated cardioprotective pathways in a similar extent. The discrepancy between our findings and previous animal and clinical studies warrant further exploration in studies designed specifically to evaluate the potential protective effects of exenatide, or of other GLP-1 agonists, such as liraglutide, in the context of cardiac surgery.


PCC 6803 lacking all group 2 σ factors. The high RNAP-SigA holoenzyme content directly induces transcription of a particular set of housekeeping genes, including ones encoding transcription and translation machineries. In accordance with upregulated transcripts, ΔsigBCDE contain more RNAPs and ribosomal subunits than the control strain. Extra RNAPs are fully active, and the RNA content of ΔsigBCDE cells is almost tripled compared to that in the control strain. Although ΔsigBCDE cells produce extra rRNAs and ribosomal proteins, functional extra ribosomes are not formed, and translation activity and protein content remained similar in ΔsigBCDE as in the control strain. The arrangement of the RNA polymerase core genes together with the ribosomal protein genes might play a role in the co-regulation of transcription and translation machineries. Sequence logos were constructed to compare promoters of those housekeeping genes that directly react to the RNAP-SigA holoenzyme content and those ones that do not. Cyanobacterial strains with engineered transcription and translation machineries might provide solutions for construction of highly efficient production platforms for biotechnical applications in the future.We show that the formation of the RNAP holoenzyme with the primary σ factor SigA increases in the Δ Recently cyanobacterial research has focused on possibilities to use cyanobacteria as bio-factories to produce valuable compounds. Lack of efficient, easily-controllable promoters has been a bottleneck in the application of cyanobacteria in biotechnology2. Although many E. coli promoters function in cyanobacteria and cyanobacterial promoters in E. coli, none of the commonly used E. coli production systems functions well in cyanobacteria. These experiences suggest that transcriptional regulation differs notably between cyanobacteria and E. coli.Cyanobacteria are eubacteria characterized by oxygenic photosynthesis. They branched early from other eubacteria and are currently found where ever light is available, from oceans to hot springs and fresh water environments to desert crust. These important primary producers are estimated to be responsible for one-third of carbon fixation on Earth3. Only the plastid encoded RNAPs of plants and algae share this arrangement4. As plastids are descendants of cyanobacteria, the splitting of β′ has obviously occurred only once in evolution. The biological reasons and consequences of β′ splitting remain to be solved. The β′ subunit bears a 600 amino acids long cyanobacteria lineage specific insertion5 whose physiological role remains unclear as well. The small ω subunit of RNAP is non-essential in cyanobacteria just like in many other eubacteria6, and it might play a regulatory role in cyanobacteria7.The RNA polymerase (RNAP) has gained specific features in the cyanobacterial lineage. In the vast majority of eubacteria, the RNAP core is composed of two α subunits and a single β, β′ and ω subunit. In the cyanobacterial lineage, the RNAP core has a unique composition with six subunits. The β′ subunit has been split in cyanobacteria, and the N-terminal part is called γ and the C-terminal part retains the name β′8. This competition is affected by numerous factors including the amount of each σ factor, affinity of different σ factors to the RNAP core, amounts and activities of anti-σ factors that prevent recruitment of a particular σ factor, and RNAP modifying factors like the small signaling molecule ppGpp that directly binds to the RNAP core and changes the recruitment efficiency of different σ factors and affect the promoter selectivity of the RNAP holoenzyme10. These processes are not yet well understood in cyanobacteria.For transcription initiation, the RNAP core recruits one of the several σ factors to form a transcription initiation competent RNAP holoenzyme. All σ factors compete for the same RNAP core13. The primary σ factor is assumed to be mainly responsible for transcription of housekeeping genes during growth. In a particular stress condition, the recruitment of one or more of the group 2 σ factors increases. Accordingly cells lacking group 2 σ factors show acclimation defects in high salt15, bright light17 or heat stresses20. In addition to stress responses, the group 2 σ factors control metabolic changes during dark-light transitions24.Cyanobacteria typically encode multiple group 2 σ factors that closely resemble the essential primary σ factor but are non-essential in standard growth conditionsSynechocystis sp. PCC 6803 strain without any functional group 2 σ factors13. The ΔsigBCDE strain is vulnerable to all tested stress conditions but grows well in standard conditions13. However, even in the standard conditions almost 20% of genes are at least two-fold up or down regulated in ΔsigBCDE compared to the control strain13. Interestingly, many genes encoding subunits of the transcriptional or translational machineries are upregulated in the ΔsigBCDE mutant. In the present paper, we further investigated the regulation of transcriptional and translational machineries in cyanobacteria. The results indicate that in the absence of competition between SigA and group 2 σ factors, more RNAP-SigA holoenzyme is formed than in the control strain. Numerous RNAP-SigA holoenzymes enhance the transcription of a particular set of housekeeping genes including those encoding transcription and translation machineries, and ΔsigBCDE produces more RNAPs and ribosomal subunits than the control strain. The extra RNAPs are fully functional, and enhanced transcription leads to a high RNA content per cell whereas extra ribosomal subunits do not form translationally active ribosomes, and ΔsigBCDE cells show similar translation activity and protein content as the control strain. Co-regulation of transcription and translation machineries as well as promoter differences between subgroups of housekeeping genes will be discussed.We have constructed a sigBCDE strain that lacks all functional group 2 σ factors grows well in standard growth conditions13. DNA microarray analysis of the transcriptome of ΔsigBCDE in the standard growth conditions revealed that many genes encoding proteins for transcription and translation machineries were up-regulated in the ΔsigBCDE strain13   Fig. . Similar CS Fig.  althoughsigBCDE than in the control strain while the amount of free SigA protein did not differ between the strains  and the Rpl1  proteins  were only detected in Fraction 19  are also typical. As the amount of translating ribosomes was similar in both strains but many housekeeping genes produce more copies of mRNA in ΔsigBCDE than in CS, the number of ribosomes reading each mRNA molecule must be lower in ΔsigBCDE than in CS.To find reason(s) for the discrepancy between the ribosomal subunit content and translation activity we further analyzed the ribosomes of Δ 19 Fig.  whereas  19 Fig. . AlthougsigBCDE but not in CS. As Fractions 1–11 did not contain full length 16S rRNA, they do not represent functional ribosomal particles. In the lightest fractions, Rps1 might be a free protein while in the other fractions it might form complexes with some other ribosomal proteins and partially degraded 16S rRNA, seen as shorter RNA species. Similarly, the Rpl1 proteins and shorter RNA fragments in Fractions 10–17 detected in the ΔsigBCDE strain might represent functionally incompetent 50S ribosomal particles , secY-adK (TU836), infA (Tu835), rpl36-rpl31 (TU833)26. Our RNA secondary structure analysis revealed possible transcription termination loops downstream of the rpl15 and secY genes. Because combining all these results turns out to be complicated, we next performed a Northern blot analysis.The est Fig. . In addiase Fig. . HoweverrpoA probe. The longest transcripts were circa 16 000 nt and could thus comprise genes from rpl3 to rpl31  were abundant together with 2700 nt, 2200 nt and 1500 nt long transcripts  and rpoB (the β subunit) genes form an operon together with the tatD gene (subunit of the twin-arginine protein export system) and the rps20 gene (a subunit of 30S ribosome), while the rpoC1 gene (the γ subunit) produces a monocistronic transcript . In standard growth conditions the RNAP-SigB holoenzyme content is low13 and thus the regulation of the sigA gene by the SigB factor is important only in special conditions like dark-light transitions. The sigA gene is located just upstream of the rrn operon25 but not co-transcribed with the rrn operon21. Currently three different promoters have been identified for the sigA gene. The sigA gene has suggested to form a transcription unit with a non-coding RNA ncr165027. A typical −10 region is found upstream of ncr1650 but the −35 region is unique . All together, these regulatory mechanisms lower the transcription activity of housekeeping genes in E. coli a lot without large changes in the σ70 content. Unlike in E. coli, the SigA content of cells drops drastically in the stationary phase in Synechocystis, whereas 6S RNA content increases only slightly28, suggesting that the dose of the primary σ factor might play a more direct role in controlling the formation of the RNAP holoenzyme in Synechocystis than in E. coli.In Δ8. In ΔsigBCDE, an elevated dose of SigA factor and a low amount of competitors result in formation of a high number of RNAP-SigA holoenzymes, over expression of many housekeeping genes and accumulation of extra RNAs. However, the growth rate of the ΔsigBCDE is not elevated13, indicating that overall transcriptional enhancement does not induce faster growth and on the other hand that accumulation of extra RNA is not harmful for Synechocystis cells. However, when the growth rate of Synechocystis changes due to changed environmental conditions, the growth rate and formation of RNAP-SigA holoenzymes often correlate. For example, in high CO2 the formation of RNAP-SigA holoenzymes is abundant and cells grow fast7 whereas nitrogen depletion causes cessation of growth and minimal formation of RNAP-SigA holoenzymes32. E. coli cells produce different amounts of RNAP depending on growth medium, and the RNAP level has been shown to be just above the level needed for maximal growth in the particular growth medium33. In E. coli, similarly as in Synechocystis, simultaneous overproduction of RNAP core and the primary σ70 factor enhances transcription of many housekeeping genes but does not enhance growth34.In eubacteria, all σ factors compete for the same RNAP core to form a transcription initiation competent RNAP-holoenzyme, different σ factors having different affinity to the coresigBCDE strain, a high content of RNAP-SigA holoenzyme induces expression of particular housekeeping genes13. We selected a collection of upregulated and non-upregulated housekeeping genes in ΔsigBCDE and compared their promoters. Upregulated Synechocystis promoters often resembled E. coli promoters with an extended −10 region35. The promoters of non-upregulated genes only contain a typical −10 region while the rest of the promoter region varies from gene to gene. Thus promoters recognized by RNAP-SigA holoenzyme in Synechocystis contain a clear −10 region but otherwise those promoters show high variability, suggesting that housekeeping genes are regulated in a complicated manner that requires other factors in addition to the RNAP holoenzyme. Increased availability of RNAP-SigA holoenzymes directly enhanced the transcription efficiency of genes with extended −10 region promoters in Synechocystis, while cyanobacterial-specific promoters of the photosynthetic machinery did not respond to the overdose of RNAP-SigA holoenzyme. Thus, our results suggest that activation of photosynthesis related genes requires more specific regulators than just a high content of RNAP-SigA holoenzyme. Demand for mechanisms strictly regulating expression of photosynthetic genes is obvious, as imbalances in the function of photosynthetic reactions potentially induces production of harmful reactive oxygen species in cyanobacteria36. We have previously shown that in the ΔrpoZ strain, a low RNAP-SigA holoenzyme content lowers especially the transcription of genes encoding the carbon concentrating and carbon fixating machineries while expression of many other housekeeping genes remain at the normal level6. Patterns of gene expression in the ΔsigBCDE and ΔrpoZ strains show that different functional groups of housekeeping genes respond differently to the dose of the RNAP-SigA holoenzyme. This might be a common phenomenon in bacteria, as a recent study showed that the dose sensitivity of the RNAP-σS holoenzyme correlates with the functional category of the σS regulated genes in E. coli37.In the ΔsigBCDE strain produces more rRNAs than CS. In E. coli activity of rRNA operons correlates with growth rate38 and the presence of thirteen highly active rRNA operons in Vibrio natriegens has been use to explain the rapid growth of this bacterium (doubling time of only 10 min)39. High rRNA content but normal growth of ΔsigBCDE shows that growth rate and rRNA content do not directly correlate in Synechocystis.The ΔE. coli40. The rrn operons of E. coli contain two promoters P1 and P2; P2 provides rather constant expression while P1 is highly induced or repressed depending on conditions38. The high transcription efficiency from the P1 promoters has been explained by their unique features that lead to formation of a scrunched open complex, thereby reducing abortive initiation41. The two identical rrn operons of Synechocystis have GTP as an initiator nucleotide and they contain only a single promoter resembling the σ70 consensus promoter with neither an extended −10 region nor a discriminator sequence typical for the P1 promoter  and the amount of the starvation induced ppGpp molecule, set the expression level of the rRNA operons in Synechocystis does contain Nus proteins that regulate Rho-dependent attenuation of the rrn operon in E. coli38, but the Rho factor is missing and thus an E. coli type attenuation mechanism cannot function in cyanobacteria. Nus proteins in Synechocystis might be involved in rRNA processing in the same way as was recently reported in E. coli44. At least rRNAs are efficiently processed in ΔsigBCDE, as only 16S, 23S and 5S rRNAs were detected and according to microarray data13 transcripts of many Nus proteins and rRNA processing enzymes were up-regulated in ΔsigBCDE.45. In accordance with that, the high rRNA content of ΔsigBCDE is accompanied with high ribosomal protein content in the mutant strain. Further studies are required to reveal the actual mechanism regulating ribosome production in Synechocystis.Ribosomal RNA content regulates the production of ribosomal proteins with a feedback mechanism in which ribosomal proteins bind strongly to rRNA and weakly to their own mRNAsigBCDE strain. Interestingly, both the rpoA operon and the rpoB-rpoC2 operon contain ribosomal protein genes in addition to RNAP genes. Actually the long rpoA cluster in Synechocystis comprises three ribosomal protein operons of E. coli -rpoA-rpl17 gene cluster suggests that the rpoA operon might play a central role for simultaneous production of transcriptional and translational machineries. The biogenesis of the RNAP core starts with the formation of the α dimer46 but the actual roles of Rps13, Rps11 and Rpl17 proteins in the formation of functional ribosomes during the complicated assembly of ribosomes (for a review see47) remains to be elucidated.Our results show that the production of both transcription and translation machineries are similarly up-regulated in the ΔsigBCDE, but translation activity and the total protein content of the control and ΔsigBCDE strains are similar. The amount of functional ribosomes in ΔsigBCDE remains similar as in the control strain, pointing to post-translational regulation of the number of active ribosomes. A possibility to engineer a cyanobacterial strain with high transcription and translation capacity without a high growth rate might offer new solutions for the use of cyanobacteria for efficient production of valuable compounds in future.Our findings about cyanobacterial regulation of transcriptional and translational machineries are summarized in Fig. Synechocystis sp. PCC 6803 (CS) and ΔsigBCDE were grown in BG-11 medium buffered with 20 mM Hepes, pH 7.5, in normal air at 32 °C under constant illumination at the photosynthetic photon flux density of 40 µmol m−2 s−1 as described earlier13. The BG-11 agar plates of ΔsigBCDE were supplemented with appropriate antibiotics but for the experiments, liquid cultures were grown without antibiotics.The glucose-tolerant strain of 730, dense cultures were diluted to OD730 = 0.4 before measurements. To determine the cell number of the CS and ΔsigBCDE cultures, the OD730 was set to 0.6 and cells were calculated with a flow cytometer BD LSRRortessa (BD Biosciences).The cell content was detected by measuring OD730 was 1.0) grown in standard conditions was harvested at 4 °C by centrifugation at 4500 g for 6 min. Then total proteins were isolated as described earlier12 and protein concentration was measured with Bio-Rad DC protein assay kit. Then 15 µg of proteins were solubilized and separated with 10% NEXT GEL® SDS-PAGE (Amresco), transferred to Immobilon-P membrane (Millipore) with Trans-Blot® (BioRad) according to the manufacturer’s instructions. The RNAP core proteins and the primary σ factor SigA were detected by Western blotting as described13. Immunoblots were quantified using a FluorChem image analyzer (Alpha Innotech Corp). As a loading control, ATPase β subunit was detected with a specific antibody (Agrisera). Three independent biological replicates were analyzed.To analyze the amounts of the RNAP subunits, 30 mL of culture ). Then 180 µL of BN isolation buffer and the pellet volume of acid-washed 150–212 µm glass beads (Sigma) were added, and cells were broken by vortexing 8 × 1 min at 4 °C. Cell debris and membranes were removed by two centrifugation steps, first at 10 000 g for 5 min and then 18 000 g for 15 min.−1, 2% glycerol, 90 mM sucrose, 2.5 mM BisTris) for 5 min on ice, and separated using 5–12% gradient gels (acrylamide:bis-acrylamide 32:1) with 4% of stacking gel using Mini protean gel system (BioRad) at 4 °C. The anode buffer was 50 mM BisTris pH 7.0 and the cathode buffer 50 mM Tricine, 15 mM BisTris pH 7.0 supplemented with 0.01% Serva G; the voltage was increased after 30 min of run from 75 V to 100 V and again after 30 min to 150 V. Then after 30 min, the cathode buffer was changed to a similar buffer without Serva G and the run was continued at 200 V until the blue color ran out of the gel. The gel was soaked in a transfer buffer  for 30 min and thereafter protein transfer and immunodetection was performed as described above. Two independent biological replicates were analyzed.To separate protein complexes, 60 μg of soluble proteins were solubilized with BN solubilization buffer (Serva G Blue 5 mg mL730 was 1) with the hot phenol method as described earlier48, and any DNA contamination was removed with TURBO DNA-freeTM kit (Ambion). The RNA content was measured with a spectrophotometer at 260 nm (Biodrop). To visualize RNA, a sample of isolated RNA from the same amount of the cells was separated on 1.2% agarose gel and stained with ethidium bromide. The DNA microarray data13 is available in GEO accession GSE69981.Total RNAs was isolated from 1-ml cell culture (OD6. Reverse transcription (RT) was performed for 900 ng RNA using SuperScript III kit (Invitrogen) according to the manufacturer’s instructions. Then 2 µL of RT reaction was used in sequent PCR amplification with Phusion HeatShock II (Thermo Scientific). The PCR primers are listed in Supplemental Table DNA-free RNA was extracted from CS as described earlier730 was 1) was supplemented with rifampicin (500 μg mL−1) and cells were collected by centrifugation after 0, 5 or 15 min of incubation in the standard growth conditions. RNA was isolated with the hot-phenol method48. Samples containing 8 μg of total RNA were denatured with the glyoxal system, separated on 1.2% agarose gel in phosphate buffer and transferred to a Hybond-N membrane48. The rpoA gene was amplified from genomic DNA using specific primers (Supplementary Table 32P-dCTP 10 mCi mL−1 (Perkin Elmer) with the Prime-a-gen labelling system (Promega) according to the manufacturer’s instructions. After prehybridization in 6xSSC, 1xDenhardt’s, 0.1% SDS, 100 μg mL−1 Herring sperm DNAS for 1 h at 67 °C, denatured probe was added and hybridized overnight at 67 °C. Then membrane was washed once with 2xSCC, 0.1% SDS at 60 °C for 15 min and once with 1xSCC, 0.1% SDS at 60 °C for 15 min and autoradiographed.The cell culture  were collected from standard conditions by centrifugation and ribosomes were isolated at 4 °C. The cell pellet was rapidly washed with 20 mM Na-EDTA, 20 mM Tris-HCl pH 8 and then with isolation buffer ). The pellet was resuspended in 2 mL of isolation buffer and the cells were broken using a French press (Constant Systems Ltd) treating the samples twice at 2.7 kPa. Cell debris was removed by centrifugation at 10 000 g for 10 min, and the supernatant was centrifuged again at 30 000 g for 30 min. The supernatant was collected and protein concentration was determined. Two mg of proteins were loaded onto the top of 5% to 45% linear sucrose density gradient in isolation buffer and the samples were centrifuged in TH-641 rotor  at 107000 g for 18 h at 4 °C. After centrifugation, 19 fractions, 620 μL of each, were collected. Three independent biological replicates were analyzed.Cells . Proteins were transfer to the membrane and immunodetected as described above. The Rps1 (AS08309) and Rpl1 (AS111738) antibodies were purchased from Agrisera.Ribosomal proteins were analyzed by western blotting. A 24-μL sample of each fraction was solubilized and proteins were separated on 12% SDS-PAGE gels with 4% stacking gel (acrylamide: bis-acrylamide 37.5:1) using ProteanTotal RNAs were isolated from each fraction. An aliquot of 300 μL of a fraction was mixed with 200 μL of RNA isolation buffer  and treated with one unit of DNaseI (Life Technologies) for 5 min and then extracted once with phenol:chlororoform (1:1) and once with chloroform. RNAs were precipitated at −20 °C overnight after addition of LiCl to a final concentration of 0.725 M and 2.5 volumes of ethanol. RNAs were collected by centrifugation at 18 000 g for 15 min and the RNA pellet was washed once with 70% ethanol and dissolved into 20 μL of water. Fifteen μL samples were separated on 1.2% agarose gels in TAE buffer and stained with ethidium bromide.35S-methionine  was added to 20 mL of cell culture (OD730 was 1) and cells were labelled for 10 or 30 min under the standard growth conditions. Cold L-Met was added to a final concentration 0.4 mg mL−1, samples were rapidly cooled and cells were harvested by centrifugation at 4500 g for 6 min at 4 °C and total proteins were isolated as described15. Proteins (60 μg) were separated by 10% NEXT GEL SDS-PAGE, transferred onto membrane and autoradiographed. Two independent biological replicates were performed.Cells were grown in standard growth conditions. Five μL of radioactive Supplementary informationDataset 1


From the viewpoint of bonding strength, the coatings with thickness below 250 µm were considered suitable for biomedical applications according to current international standards. A mechanical model based on quantized fracture mechanics allowed estimating the fracture toughness of the coating on the basis of the experimental data from tensile tests. The critical strain energy release rate was also found to decrease from 1.86 to 0.10 J/m2 with the increase of coating thickness, which therefore plays a key role in determining the mechanical properties of the materials.Deposition of bioactive glass or ceramic coatings on the outer surface of joint prostheses is a valuable strategy to improve the osteointegration of implants and is typically produced using biocompatible but non-bioactive materials. Quantifying the coating–implant adhesion in terms of bonding strength and toughness is still a challenge to biomaterials scientists. In this work, wollastonite (CaSiO Deposition of glass or ceramic coatings on orthopedic and dental implants is useful to impart better biocompatibility and bone-bonding properties  to the uPlasma-sprayed hydroxyapatite coatings have been used for many years on titanium implants in orthopedic and dental surgery ; howeverThe literature on biomedical glass coatings is mainly focused on both the search for effective deposition methods, which evolved from conventional enameling  to elect2–CaO–Na2O–Al2O3 base glass) and substrate  is quantitatively studied from this viewpoint. Specifically, the bonding strength of the coatings was experimentally assessed by performing tensile tests, and the fracture toughness was then determined by applying a properly derived model based on quantized fracture mechanics.In this work, it is studied how thickness can affect the mechanical performance of silicate glass–ceramic coatings deposited onto alumina. To the best of the author’s knowledge, it is the first time that such a combination of coating material (SiO2–34CaO–6Na2O–3Al2O3 wt.%), previously developed for biomedical applications [2, CaCO3, Na2CO3, and Al2O3, all purchased from Sigma-Aldrich, St. Louis, MO, USA) were homogenously mixed for 30 min in an orbital shaker; then, the blend of powders was transferred to a platinum crucible, heated to 1150 °C (heating rate 10 °C/min) in an electrically heated furnace, and melted for 1 h in air. The melt was quenched into deionized water to obtain a frit that was ball-milled  and sieved by a stainless-steel sieve  to a final particle size below 32 μm.The starting silicate glass  by gravity-guided deposition after suspending proper amounts of glass powder in ethanol. The glass particles were left to deposit on the substrates overnight; then, the containers hosting the materials were placed in an oven at 90 °C for 6 h to remove the excess ethanol and allow complete dying of the “greens”. The samples were then thermally treated in an electrically heated furnace at 1000 °C for 3 h (heating rate 5 °C/min) in order to allow glass particle sintering and coating consolidation.Glass coatings of different nominal thicknesses  were manufactured on flat high-purity alumina tiles , crystallization onset (Tx), crystallization peak (Tp), and melting (Tm). Specifically, glass powder was analyzed by using a DTA 404 PC instrument  in the temperature range of 25 to 1400 °C with a heating rate of 10 °C/min. After being introduced into a platinum crucible provided by the instrument manufacturer, 50 mg of glass underwent the thermal cycle; an equal amount of high-purity Al2O3 powder was put in the reference crucible. Standard calibration procedure and baseline corrections were performed.Differential thermal analysis (DTA) was performed on the glass powder to determine the characteristic temperatures of the material, i.e., glass transition temperature (Thttp://pcpdfwin.updatestar.com).Sintered coatings underwent X-ray diffraction (XRD) in the 2θ-range of 10 to 60° by using a X’Pert Pro PW3040/60 diffractometer  operating at 40 kV, 30 mA with Cu Kα incident radiation (wavelength 0.15405 nm), step size 0.02°, and counting time 1 s per step. Crystalline phase assignment was performed by using X’Pert HighScore software 2.2b  equipped with the PCPDFWIN database  at an accelerating voltage of 15 kV. The thickness of the coating was measured directly on the SEM images by making use of an image-analysis software provided by the manufacturer; the results were expressed as average ± standard deviation of three measurements per each sample. Compositional analysis was performed by energy dispersive spectroscopy (EDS); the probe was included in the SEM equipment.The coating cross sections and the substrate–coating interface were also inspected by scanning electron microscopy (SEM). For this purpose, the samples were embedded in epoxy resin , cut by a diamond wheel , and polished using #600 to #4000 SiC grit paper; the resulting cross sections were sputter-coated with silver and analyzed by field-emission SEM , according to an experimental procedure described elsewhere [σ) was calculated as the maximum load (F) per unit cross-sectional area (A):The tensile (bonding) strength was measured by applying tensile loads to pull the coating apart, following the relevant ASTM standard . Before lsewhere . The bonPull-out tests were performed on five specimens per each of the six sample batches having different nominal thickness; the results were expressed as average ± standard deviation.U is the strain energy, and W is the work done by the external force F. Considering our case, U and W can be calculated as:k1 and k2 are the stiffness of alumina tile and coating, respectively, before crack propagation.An estimation of the coating toughness was obtained from the values of tensile strength (see Equation (1)) by implementing an appropriate model, based on fracture mechanics, which was derived as follows. The theory of linear elastic fracture mechanics states that the total potential energy of a system can be expressed as:Hence, after combining Equations (2)–(4), it is obtained:ICG is the fracture energy (per unit area created) of the material.Griffith’s energy criterion states that crack propagation occurs when the variation of the total potential energy The recent theory of quantized fracture mechanics extends the Griffith’s criterion to discrete cracks: thus, after substituting differentials with finite differences GIC=−There are three possible failure modes occurring when the coatings undergo pull-out tests: in fact, the fracture can be (i) totally adhesive, (ii) totally cohesive, or (iii) mixed.Considering the case (i), it is ideally assumed that the failure occurs at the coating–alumina interface; therefore, the variation of the total potential energy is:E1 (=400 GPa) and E2 (=90 GPa [t1 (=1 mm) and t2 the corresponding thicknesses.The compliances in Equation (7) can be expressed as (=90 GPa ) are theThus, Equations (7) can be rewritten as:IG,12 can be expressed as:Combining Equations (6) and (8), the energy release rate Hence, the delamination  strength can be expressed as:Considering the case (ii), it is ideally assumed that the failure occurs entirely in the coating; this means that the tensile strength of the coating material (= 47 MPa ) is loweA simple visual inspection of the fracture surfaces after tensile tests revealed that there was a coexistence of the two failure modes (i) and (ii) ). Therefore, the critical stress is assumed to be predicted by a mean field approach as:After combining Equations (10) and (11), it is finally obtained:2 and the significant presence of Al2O3 resulted in an almost inert behavior with almost no apatite-forming capability in contact with biological fluids, thereby preventing the formation of a tight bond to bone. However, the material was found highly suitable to produce porous orbital implants [The silicate glass used in this work was initially proposed for potential usage in the field of bone regeneration and substitution . This coimplants , which mimplants , and mulimplants .g = 685 °C (inflection point), Tx = 800 °C , Tp = 855 °C (exothermic peak), and Tm = 1155 °C (endothermic peak).The characteristic temperatures of the glass, determined from the DTA plot , were Tg3, PDF code no. 00-027-0088) when the material was thermally treated at 1000 °C is consistent with the results from thermal analysis, showing that crystallization started at 855 °C. The XRD results were also in agreement with those obtained on the same glass system after heat treatment above 900 °C [The coatings underwent devitrification upon thermal treatment, as shown in e 900 °C . No probe 900 °C .−6 °C−1 [−6 °C−1).Analysis of the cross section revealed that the interface between coating and alumina tile was flawless without any apparent interfacial crack or delamination in intact samples a. Howeve−6 °C−1 ) and aluThe thicknesses of the coatings, reported in 3 crystals , exhibiting a typical acicular shape, were embedded in a grey matrix in which high amounts of Al and Na could be found, too, along with low Ca content , corresponding to the residual glass. These results are in accordance with the XRD pattern (https://imagej.nih.gov/ij/index.html) to obtain a rough quantification of crystalline and glassy phase proportions in the glass–ceramic material: the white regions corresponding to CaSiO3 crystals were estimated to be about 70% of the total cross-sectional area of the coating.The SEM micrograph shown in  pattern , where a2 also confirmed the good fitting of the interpolating function with the experimental data.The results from the tensile tests showed that the bonding strength decreased as the coating thickness increased . Data inNo specific international standard exists about the minimum bonding strength recommended for biomedical glass coatings; however, a tensile strength of 15 MPa is prescribed by ISO 13779 for hydroxyapatite coatings on surgical implants . This reICG,12 value can be interpreted as an estimation of fracture toughness [Analysis of the fracture surfaces  confirmeoughness , this me2–CaO–Na2O–P2O5–MgO glass coatings by pulsed laser deposition on Ti6Al4V alloy and observed that thick coatings showed poorer adhesion to the substrate. Matinmanesh et al. [2–CaO–Na2O–P2O5–ZnO coatings on Ti6Al4V alloy, too, and demonstrated that increments in the coating thickness led to higher residual stresses that tended to increase the “crack driving force”. This interesting explanation can be extended to the present work. The residual stresses in the coating contributed to the release of the stored strain energy during the tensile test and, therefore, less energy from the applied loads was needed to reach the critical value required for crack propagation. In other words, lower tensile loads were required to overcome the resistance of thicker coatings against cracking because of higher residual stresses.Gomez-Vega et al. ,26 claimh et al.  reported2 [Wollastonite-containing glass–ceramic coatings (thickness 140 µm) deposited on alumina by airbrushing in a previous work exhibited a fracture toughness of 0.8 J/m2 , which i2. A significant drop of ICG,12 values accompanied by a decrease of dA  relationship between fracture toughness and thickness of the coating , as confse of Ad  could beThe bonding strength and critical strain energy release rate of wollastonite-containing glass–ceramic coatings were found to decrease as the coating thickness increased, i.e., thinner coatings proved to have higher resistance against fracture than thicker ones. These relationships were quantified by combining experimental data from tensile tests on the coatings and a model based on quantized fracture mechanics. The obtained functions are very valuable to link the coating thickness, which can be tailored at the design and manufacturing stages, with those key mechanical characteristics. Specifically, it could be predicted what is the maximum thickness allowed so that the coating can exhibit the mechanical characteristics required for a given application. Therefore, the coating thickness should be carefully taken into account in order to optimize the material performance and design. The results also suggest that, ideally, the coating should be as thin as possible for increasing the mechanical performance; thus, further improvements are forecast if the coating thickness decreases below the minimum value (50 µm) considered in this study. The same methodological approach followed to estimate toughness could also be applied to different glass or ceramic coatings based on other compositions of biomedical interest.