Carbon fiber-reinforced plastic (CFRP) is widely used for structural members of transportation vehicles such as automobile, aircraft, or spacecraft, utilizing its excellent specific strength and specific rigidity in contrast with the metal. Short carbon fiber composite materials are receiving a lot of attentions because of their excellent moldability and productivity, however they show complicated behaviors in fatigue fracture due to the random fibers orientation. In this study, thermoelastic stress analysis (TSA) using an infrared thermography was applied to evaluate fatigue damage in short carbon fiber composites. The distribution of the thermoelastic temperature change was measured during the fatigue test, as well as the phase difference between the thermoelastic temperature change and applied loading signal. Evolution of fatigue damage was detected from the distribution of thermoelastic temperature change according to the thermoelastic damage analysis (TDA) procedure. It was also found that fatigue damage evolution was more clearly detected than before by the newly developed thermoelastic phase damage analysis (TPDA) in which damaged area was emphasized in the differential phase delay images utilizing the property that carbon fiber shows opposite phase thermoelastic temperature change. Carbon fiber reinforced plastic (CFRP) has been widely employed for the primary structural members of transportation vehicles such as automobile, aircraft, or spacecraft, utilizing its excellent specific strength and specific rigidity in contrast with the metal. Short carbon fiber composite materials have been receiving a lot of attentions because of their excellent moldability and productivity. However, they show complicated behaviors in fatigue fracture due to their random fibers orientation. Therefore, effective nondestructive evaluation techniques are required for detecting and measuring various types of damages—such as fiber breakage, matrix cracking, and delamination—during fatigue fracture. Nondestructive evaluation techniques using infrared thermography, i.e., thermographic NDT has been effectively employed for the detection of delamination defects in fiber reinforced plastics. The thermographic NDT techniques based on thermal insulation effect by the delamination defect have been applied to the nondestructive testing of composite materials. The combination of active pulse heating and subsequent transient temperature measurement was effectively employed for sensitive detection of delamination damage in composites. Avdelidis et al. ,2 reviewIn contrast with these thermal insulation techniques, damage evaluation techniques based on the thermoelastic stress analysis (TSA) using infrared thermography were examined for CFRP composites. TSA is a well-established experimental technique for the evaluation of the stress field in isotropic materials, and it has come into widespread use in the industry as an effective, experimental, full-field stress measurement technique ,10,11,12Thermoelasticity for orthotropic materials have been studied by many researchers and the TSA technique has been employed as a powerful tool for evaluating impact or fatigue damage in composite materials and structures ,20,21,22The present authors  investigTE) to the sum of the changes in the principal stresses (∆σ) under cyclic variable loading as follows.Dynamic stress change cause a very small temperature change under adiabatic conditions in a solid. This phenomenon is known as the thermoelastic effect and is described by Lord Kelvin’s equation , which rα is the coefficient of thermal expansion, ρ is the mass density, pC is the specific heat at constant pressure and T is the absolute temperature. The coefficient k is called thermoelastic constant. The sum of the changes in the principal stresses (∆σ) is obtained by measuring the temperature change (∆TE) using infrared thermography.Here, As thermoelastic temperature changes are very small and sometimes hidden by the thermal noise of the infrared camera, lock-in infrared thermography using reference signals synchronized with the stress changes is commonly employed to improve the accuracy of stress measurements. The TSA technique uses a lock-in algorithm with a reference-loading signal extracted from the load cell or strain gauge to improve the signal–noise ratio.k, the thermoelastic temperature change shows opposite phase waveform against the loading waveform. In this study, the value of the phase difference ∆θE was defined as the difference in phase between the thermoelastic temperature change and the loading signal as shown in the figure. In this case, phase difference ∆θE is 180 deg. In this study, thermoelastic temperature measurement was conducted for the short carbon fiber reinforced plastics, and the thermoelastic temperature change ∆TE and the phase difference ∆θE was obtained from experimental data.When a tension and compression loading in sinusoidal waveform as shown in Configurations of the CFRP specimen employed in this study are shown in f of 7 Hz and a stress ratio R = 0.1 was applied to the specimen by an electrohydraulic fatigue testing machine. Microscopic visible images on the specimen surface and side surface were taken by optical microscope. The temperature change on the specimen surface was measured by infrared thermography with an MCT array detector (CEDIP Inc. (FLIR Systems Inc.), Croissy Beaubourg, Fracnce , Titanium530L). The specifications and setting of this infrared camera are listed in TE and the phase difference ∆θE were obtained from experimental data as shown in the forgoing paragraph.Cyclic-axis sinusoidal waveform loading with a frequency σmax = 100 MPa. The fiber orientation angle φf is defined as shown in φf is equal to 0 degrees when a fiber bundle is oriented in parallel with the loading axis. The distribution of the fiber orientation angle φf was measured after the thermoelastic stress measurement as described below: (1) surface thin resin layer was removed by polishing for exposing carbon fiber bundles; (2) optical image of the surface carbon fiber bundles was taken by digital camera; (3) fiber orientation angle φf was determined by image processing program developed by Enomae [Before conducting fatigue test, the effect of the fiber orientation angle on the thermoelastic temperature change was experimentally investigated using a CFRP specimen without notches. Measurement of the thermoelastic temperature change was conducted under the applied maximum stress y Enomae .TE obtained for CFRP specimen without notches are shown in φf is shown in TE measured in the area where φf is around 90° takes higher values compared with those measured in the area where φf is around 0°. Furthermore, the relationship between the fiber orientation angle φf and the phase difference ∆θE is shown in θE takes the value of 180° where the fiber orientation angles are around 90°. On the other hand, ∆θE takes 0° where φf is around 0°.Obtained optical image of the sample surface, distribution of fiber orientation angles, and thermoelastic temperature change ∆k of unidirectional CFRP takes negative value. It is supposed that carbon fibers mainly shared the applied axial load in the region where the orientation angles of the carbon fiber bundles are 0° . Therefore, the thermoelastic temperature change showed a coordinate-phase waveform with that of applied loading.It was reported by Sugimoto and Ishikawa  that theσmax = 180 MPa. As described in the introduction, Uenoya et al. [TE on the fracture part was decreasing with the increasing loading cycles. And the decreasing area of ∆TE was expanded in the transverse direction of the specimen. This indicates that fatigue damage can be detected from the local stress change caused by the change in load sharing conditions due to the damage evolution.Fatigue test was conducted for the short carbon fiber reinforced plastic specimen with circular notches under the applied maximum stress a et al.  developeθE obtained at 200; 10,000; 30,000; 40,000; and 44,000 cycles are shown in θE = 0° means that the thermoelastic effect of carbon fiber bundles is predominant, on the other hand ∆θE = 180° means that the thermoelastic effect of matrix resin is predominant. The change in phase values indicates the change of load sharing conditions between resin and carbon fiber bundles due to the fatigue damage evolution. We thought that high-sensitivity early fatigue damage detection technique can be developed by utilizing the change in phase difference ∆θE; we propose a thermoelastic phase damage analysis (TPDA). In TPDA a differential phase delay distribution image is generated by subtracting a phase delay image obtained at certain loading cycle from a reference initial phase image. In this study the reference initial phase delay image was set at 200 cycles. The obtained TPDA images at 10,000; 30,000; 40,000; and 44,000 cycles are shown in Distribution of the phase difference ∆θE = 0° means that the thermoelastic effect of carbon fiber bundles is predominant, on the other hand ∆θE = 180° means that the thermoelastic effect of matrix resin is predominant. The change in phase values indicates the change of load sharing conditions between resin and carbon fiber bundles due to the evolution of delamination damage.In this experiment, visible images on the side surfaces of the CFRP specimen were taken by digital microscope. Microscopic visible images taken at 30,000; 35,000; and 40,000 loading cycles are shown in (1)TE and the fiber orientation angle φf that ∆TE in the area where φf = 90° takes higher value compared with that in the area where φf = 0°.It was found from the relationship between the thermoelastic temperature change ∆(2)φf and the phase difference ∆θE that ∆θE takes 180° in the area where φf = 90°. On the other hand, ∆θE takes 0° where φf = 0°. This is due to the negative thermoelastic constant of carbon fibers and the load sharing condition between resin and carbon fibers.It was also found from the relationship between the fiber orientation angle (3)Fatigue damage was evaluated according to the conventional TDA procedure as well as the newly developed phase-delay based damage characterizing technique “thermoelastic phase damage analysis (TPDA)”. It was found from TPDA images that the significant change of phase values from 0 to 180° (indicating the change in load sharing condition between resin and carbon fibers due to fatigue damage evolution) was found in the region where the structural fracture was detected.In this study, TSA using an infrared thermography was applied to the evaluation of fatigue damage in short carbon fiber composites. In addition to the conventional thermoelastic damage analysis, the phase difference between the thermoelastic temperature change and the applied loading signal was measured for short carbon fiber reinforced plastics. Obtained results are summarized as follows:


In the strong-field photoemission from atoms, molecules, and surfaces, the fastest electrons emerge from tunneling and subsequent field-driven recollision, followed by elastic backscattering. This rescattering picture is central to attosecond science and enables control of the electron’s trajectory via the sub-cycle evolution of the laser electric field. Here we reveal a so far unexplored route for waveform-controlled electron acceleration emerging from forward rescattering in resonant plasmonic systems. We studied plasmon-enhanced photoemission from silver clusters and found that the directional acceleration can be controlled up to high kinetic energy with the relative phase of a two-color laser field. Our analysis reveals that the cluster’s plasmonic near-field establishes a sub-cycle directional gate that enables the selective acceleration. The identified generic mechanism offers robust attosecond control of the electron acceleration at plasmonic nanostructures, opening perspectives for laser-based sources of attosecond electron pulses. Accelerating electrons to high energy and controlling their properties on ultrafast timescales is challenging. Here the authors show controlled acceleration of electron bunches using forward scattering in the resonantly enhanced polarization field of silver clusters driven by a phase-tuned two-color laser field. Moreover, the recollision concept provides a powerful framework for controlling the spectral8, directional10, and temporal properties11 of the laser-driven electrons.When a bound electron is liberated by a strong laser field, it may escape or return to the residual potential to recombine or rescatter. This field-driven recollision is at the heart of strong-field scienceUp cutoff (with Up the ponderomotive energy)12, while forward rescattering only produces so-called low-energy structures15. These slow forward rescattered electrons enable electron holography through their interference with direct electrons16. Still, as for atoms and molecules, elastic backscattering also governs the near-field enhanced high-energy photoemission from the surface of nanotips, nanospheres, and plasmonic nanostructures19. For these extended systems, forward rescattering may even be quenched completely by electron scattering in the medium. As elastic backscattering dominates the fast electron emission from point-like targets and surfaces, similar protocols can be employed to achieve waveform control.In atomic photoemission, i.e., for emission from nearly point-like potentials, elastic backscattering forms the rescattering plateau in the electron energy spectra with the well-known 10 21. The underlying concept, being also the key trick in laser grating accelerators22, is that the electron’s travel distance per optical cycle can match the scale of the system’s static or induced potential. In consequence, optical waveform control of scale-matched forward rescattering should be possible and can be studied using clusters as model systems. The ramifications of such control would be of general interest for electron acceleration based on plasmonic nanostructures.In a finite system like a cluster Fig. , however23 facilitates the formation of nanometer-sized plasmas that emit X-rays24, energetic25, and highly charged ions26, as well as fast electrons. The emerging deep cluster potential strongly confines plasma electrons and permits robust plasmonic resonance excitation after appropriate cluster expansion27. Besides high absorption, resonance excitation also triggers particularly energetic electron emission along the polarization axis that evidences field-driven acceleration28. According to theory, the acceleration proceeds in a single laser cycle during the electron’s final transit through the cluster, reflecting the above mentioned forward rescattering with matched scales. Both direct laser field-driven acceleration20 and acceleration by the cluster polarization field have been put forward to explain the enhanced energy gain21. In both cases, the forward rescattering process should be sensitive to the field evolution in the relevant laser cycle. An experimental demonstration of this dependence, however, was still lacking.The ability of clusters to efficiently absorb intense laser lightIn the current study we observe the waveform-dependent electron emission from resonantly driven silver clusters. We find directional control with the phase of a bichromatic laser field, identify the underlying near-field mediated gating mechanism, and describe its implications for the control of attosecond electron pulses.ω/2ω probe pulse (Methods), containing a dominant NIR fundamental and a weaker second harmonic component (γ2 = Iω2/Iω ≈ 0.2). The sub-cycle waveform of the linearly polarized ω/2ω probe field φ. Direction-resolved energy spectra are recorded along the polarization axis with a stereo electron spectrometer (Methods). For resonant ω/2ω-probe excitation, we find strongly enhanced and highly phase-dependent directional acceleration, characterized by up–down emission asymmetries A up to ±0.7  denotes the value of the relative phase φ with maximal upward emission and is spectrally nearly flat , exceeding the energies found for ω/2ω excitation of Ar carrier gas (probe pulse only) by more than an order of magnitude  pump pulse Fig. ; Methods0.7 Fig. . The retlat Fig. . The obsude Fig. . The atotra Fig.  and enabtra Fig. .The phase-controlled acceleration is analyzed with semiclassical molecular dynamics (MD) simulations (Methods). Up to a small offset in the asymmetry phase Fig.  that is φ = 0, where the asymmetry becomes most apparent, reveals the main physics  = 0. The most energetic electrons escape in a short period of resonant coupling in the trailing edge of the ω/2ω pulse , where i is the particle index, reveals energy gains approaching 1 keV in a single fundamental laser cycle  is the potential created by all other charges. The associated single-particle energy gain rateA representative MD run with relative phase ics Fig. . For eaclse Fig. , underlilse Fig. . The attlse Fig. . Note thcle Fig. . Here V = Vpol(x)p(t). Here p(t) = P0[cos(ωt) + γpol cos(2ωt + φpol)] is the two-color dipole moment  is the static polarization potential. The laser field creating the polarization is neglected.The universal nature of the phase-controlled acceleration and its implications for forming or modifying attosecond electron bunches is highlighted by a one-dimensional nanoaccelerator model. Therein, electrons are considered to cross a dynamically polarized sphere Fig.  with potent Fig.  with relP0, the energy gain can be approximated using unperturbed drift trajectories x(t) = (t − ttrans)vd with drift velocity vd. Focussing on the timing, the energy gain is maximal for transits centered in the respective gate  the laser and dipole relative phases are equal such that the requirement for maximal asymmetry, φpol = 0, is met for φ = 0. Deviations from this value in the experimental data and MD results  as confirmed by particle mass spectrometry34 and TEM analyses. The cluster beam has a diameter of 2 mm when entering the interaction region. The experiments are performed in the limit, where only a single cluster is excited in each laser shot. The stereo time-of-flight (TOF) spectrometer contains two opposite, field-free and magnetically shielded 0.5 m drift tubes  into the interaction region, where it intersects the molecular beam. The laser focus is placed 3.5 mm behind the molecular beam to reduce phase averaging due to the Gouy phase shift.NIR laser pulses  from a 1 kHz laser system  are split into pump and probe pulses using a Mach–Zehnder interferometer Fig. . The 40035. Atomic tunneling and electron impact ionization events are treated via effective rates. The classical propagation of plasma particles uses a regularized Coulomb interaction that is matched to atomic ionization energies and impedes classical electron–ion recombination below the atomic energy levels. The force evaluation in the gridless code is accelerated by exploiting massively parallel processing. Local field effects such as field enhancement and the depression of ionization potentials due to plasma screening are included in the evaluation of tunneling and electron impact ionization, respectively. Spherical silver clusters are initialized with fcc structure and a single active conduction electron. The conduction electrons were initialized with 3 eV kinetic energy, which roughly reflects the average valence electron kinetic energy in the bulk given by 3/5EF, where EF = 5.5 eV is the Fermi energy of silver. The simulations cover the full dynamics resulting from the excitation with pump and probe pulses. For the considered cluster sizes, the laser field can be described in dipole approximation. Electromagnetic effects are neglected. Within the resonant pump-probe excitation on average 8.2 additional electrons were removed from each atom, leading to a total inner ionization of qii = 9.2 per atom. The electron emission into the continuum almost exclusively takes place in the probe pulse, reaching a total outer ionization degree of qoi = 3.1 for the considered parameters.The waveform-dependent electron acceleration in the clusters is calculated by hybrid MD simulations that employ a microscopic classical treatment of the electron and ion dynamics in the laser-driven nanoplasmami and qi are the mass and charge of the respective plasma particle, V is the instantaneous Coulomb potential created by all other charges at the position ri of the considered actual particle. Further, from Eq. (Within the classical propagation, particle trajectories are integrated according to Newton’s equation of motionmentclass2pt{minimAll measured data used in this study as well as the custom parts of the simulation code are available on request from the corresponding authors.Peer Review File


However, several microorganisms can bioconvert TeO32− into the less toxic form of elemental tellurium (Te0). Here, Rhodococcus aetherivorans BCP1 resting (non-growing) cells showed the proficiency to produce tellurium-based nanoparticles (NPs) and nanorods (NRs) through the bioconversion of TeO32−, depending on the oxyanion initial concentration and time of cellular incubation. Te-nanostructures initially appeared in the cytoplasm of BCP1 cells as spherical NPs, which, as the exposure time increased, were converted into NRs. This observation suggested the existence of an intracellular mechanism of TeNRs assembly and growth that resembled the chemical surfactant-assisted process for NRs synthesis. The TeNRs produced by the BCP1 strain showed an average length (>700 nm) almost doubled compared to those observed in other studies. Further, the biogenic TeNRs displayed a regular single-crystalline structure typically obtained for those chemically synthesized. The chemical-physical characterization of the biogenic TeNRs reflected their thermodynamic stability that is likely derived from amphiphilic biomolecules present in the organic layer surrounding the NRs. Finally, the biogenic TeNRs extract showed good electrical conductivity. Thus, these findings support the suitability of this strain as eco-friendly biocatalyst to produce high quality tellurium-based nanomaterials exploitable for technological purposes.Tellurite (TeO The anthropogenic misuse of Te-compounds in several areas of application 5 has led to an increased presence of several forms of Te in the environment, namely: inorganic telluride (Te2), the oxyanions tellurite (TeO32−) and tellurate (TeO42-), and the organic dimethyl telluride (CH3TeCH3)6. Among these Te forms, TeO32− is recognized as a soluble and hazardous pollutant, which can be found highly concentrated in soils and waters near by waste discharge sites of manufacturing and processing facilities7. Although TeO32− exerts its toxicity at concentrations as low as 1 μg mL−1 (4 μM)5 towards both prokaryotes and eukaryotes6, over the past 30 years mainly anaerobic or facultative anaerobic bacteria were investigated for their ability to bioconvert TeO32− 9, while much less is known about the bioconversion potential of aerobic bacterial strains towards these oxyanions12. Regardless of the bacterial strain investigated, a common feature reported by several authors, is that TeO32− bioconverting bacteria produces black precipitates within and/or outside the cells14. Indeed, the early work of Morton and Anderson (1941) observed needle-like crystals within and outside Corynebacterium diphtheriae cells grown on Chocolate Tellurite agar13, while Tucker and colleagues (1962) reported X-Ray diffraction analysis of Te crystalline nature of the black precipitates produced by Streptococcus fecalis N8311. Recently, these Te-crystals were recognized as nanosized structures generated by microorganisms as product of metal(loid) bioconversion16, which can be exploited to develop eco-friendly and cost-effective methods to synthesize valuable metalloid nanomaterials17. Indeed, the advantage of a microbial approach as compared to a synthetic procedure would be the abandonment of toxic chemicals, avoiding the formation of hazardous waste, and the use of extreme system conditions , which determine the emergence of safety concerns17.The chalcogen Tellurium (Te) is a natural rare element of the Earth crustRhodococcus genus have been investigated due to their environmental robustness and persistence18, with the characteristic of resisting harsh growth conditions20. In a previous study, we reported the ability of Rhodococcus aetherivorans BCP1 to cope with high concentrations of TeO32−, as well as its proficiency to bioconvert these oxyanions into the less toxic Te0, generating thermodynamically stable nanostructures21. Here, based on our prior findings, we further explored the strain BCP1 under metabolically active, yet resting (non-growing) cells. Conditions using these cells were optimized for the biotic conversion of TeO32− and to enhance the chemical-physical characteristics of the biogenic Te-nanomaterial produced. We investigated key parameters such as size, shape, and crystalline nature of the Te-nanostructures biosynthesized by BCP1, and we provided evidence for the presence of amphiphilic biomolecules in the organic layer surrounding the biogenic TeNRs, which might play a crucial role directing their growth and stabilizing them. Hence, we proposed a mechanism of assembly, growth and formation of the intracellularly generated TeNRs, whose electrical properties were evaluated as proof-of-concept of the suitability of this nanomaterial for future electronic applications.In this regard, among the strictly aerobic bacterial strains suitable as cell factories for nanotechnology purposes, those belonging to the 22 is now recognized as a valuable approach to develop green-synthesis strategies to produce unique nanoscale materials23. In our previous study, the capability of BCP1 cells grown aerobically in the presence of TeO32− to biosynthesize TeNRs as product of TeO32− bioconversion was observed21. In our research exploring different physiological conditions to optimize TeNRs production, we discovered that BCP1 resting cells had a greater performance to produce extremely long TeNRs as compared to actively growing cultures21. Indeed, although TeO32− exposure caused a certain level of cell death directly proportional to the initial concentration of oxyanions . BCP1’s tolerance towards this oxyanion was further highlighted by its capability to remove 28 ± 4% (corresponding to 280 ± 40 μg mL−1) when exposed to 1000 μg mL−1 TeO32− over 16 h , 23.4 ± 0.7 μg mL−1 h−1 (500 μg mL−1) and 36 ± 3.0 μg mL−1 h−1 (1000 μg mL−1)  Fig. . Finally32− was coupled to its proficiency to generate intracellular Te-nanostructures in the form of NPs and NRs in all experimental conditions tested  produced primarily TeNPs  both TeNPs and TeNRs were detected within the cells . Shard-like NPs were also detected along with TeNRs within Te-nanostructure extracts isolated from BCP1 cells exposed for either 0.5 or 1 h to 1000 μg mL−1 of TeO32− 8, Bacillus selenitireducens (200 nm)25 and Shewanella oneidensis MR-1 (100–200 nm)15.The measurement of the average length and diameter of TeNRs was evaluated as function of both TeOion Fig.  and S2. 0 tendency to form 1D nanostructures relies on the high thermodynamic stability of trigonal tellurium (t-Te), which is responsible for the anisotropic growth of Te-nanocrystallinities along one axis26. In this respect, the biogenic TeNRs generated byBCP1 were analyzed through HR-TEM imaging and SAED and revealed individual and regular NRs without any defects or dislocations along the longitudinal c-axis, indicating their uniform and single-crystalline structure ]27. Further, the TeNR ED pattern was indexed as pure t-Te phase with calculated lattice constants a = 4.38 Å and c = 5.83 Å .Teure Fig. . The ele9 Å Fig.  was cons3 Å Fig. , whose v32−. According to the established chemical models of TeNRs synthesis29, the formation of 1D nanostructures is preceded by the generation of NPs generally featured by Te in amorphous state (a-Te), conferring them a high surface energy and hence thermodynamic instability. Thus, TeNPs rapidly dissolve and the available Te0 atoms organize themselves depositing as t-Te in one direction forming NRs through a ripening process31. The kinetics of this event is directly dependent on the concentration of TeO32− precursor supplied, resulting in a faster dissolution of TeNPs as the initial amount of oxyanion increased31. The entire process resulted to be emphasized in BCP1, as the biotic conversion of TeO32− occurred in the cytoplasm, leading to a large number of Te0 atoms confined in the small cellular volume and available to produce TeNRs. Indeed, considering that the bioconversion rate of TeO32− increased as function of the initial oxyanion concentration  they generated both TeNPs and TeNRs at this early time point  capable of specifically binding to the hydrophobic moieties of amphiphilic molecules in the extract, leading to a change in fluorescence37, as the unbound tracer molecules are quenched in water38. Indeed, a fluorescent emission peak at 507 nm was detected for the TeNRs extract (DiOC18(3)-TeNRs extract), which was comparable to that of the lipophilic tracer dissolved in methanol (DiOC18(3)-methanol; 501 nm) -TeNRs extract and DiOC18(3)-methanol  showed emission and excitation fluorescence peaks at the same wavelengths compared to those of DiOC18(3)-TeNRs extract -methanol as compared to those obtained for both DiOC18(3)-TeNRs extract and DiOC18(3)-extruded amphiphilic biomolecules, which were similar to that of DiOC18(3)-extruded POPC liposomes  Fig. . These rnol Fig. . To undenol Fig. , indicatact Fig. . Additio40. Based on these evidences, a mechanism explaining the assembly and growth of TeNRs occurring within BCP1 resting cells is proposed in Fig. Thus, the fluorescence spectra and the FCS analyses strongly suggested that the amphiphilic biomolecules present within the aqueous biogenic TeNRs extract behaved as non-ionic surfactants (POPC), which have been previously utilized as driving force and stabilizers for chemically synthesized NRsp-type semiconductor41, it exhibits high photoconductivity, piezoelectricity, thermoelectricity and non-linear optical response properties43, allowing the use of Te-based nanomaterials as optoelectronic, thermoelectric, piezoelectric devices, as well as gas sensors and infrared detectors48. Thus, we explored the conductive properties of the biogenic TeNRs extract, measuring its resistance (R) through the Four Probe technique49. The biogenic TeNRs extract air dried on the silicon support gave a low resistance value (R = 8 ± 1 Ω), compared to that of the silicon chip itself (R = 281 ± 7 Ω), and the isolated amphiphilic molecules (R = 145 ± 2 Ω). These values corresponded to an electrical conductivity (σ) of 3.0 ± 0.5, 0.08 ± 0.002 and 0.16 ± 0.02S m−1, respectively. Hence, TeNRs within the extract were shown to be electrically conductive, approaching the electrical conductivity values of those chemically synthesized, with σ ranging between 8 and 10S m−151.Since Te is a well-known narrow band-gap Rhodococcus aetherivorans BCP1 strain to tolerate very high concentrations of the toxic oxyanion TeO32− under the physiological state of resting cells as compared to those actively growing21. Although the biotic conversion of TeO32− led to the intracellular production of different tellurium nanomaterial morphologies at early time points, the main nanostructure biosynthesized was TeNRs, whose average length was impressively long as compared to TeNRs reported in the literature so far. Moreover, the biogenic TeNRs showed a single-crystalline structure resembling those chemically synthesized, while the morphological changes of biogenic Te-nanostructures and the unchanged average diameter of the TeNRs suggested a specific mechanism of their assembly and confined growth within BCP1 cells, which might be assisted by the co-produced amphiphilic biomolecules from this Rhodococcus strain. Finally, the biogenic TeNRs extract showed to be electrically conductive, approaching those chemically produced and, therefore, underlining the suitability of this strain as an eco-friendly cell factory exploitable to synthesize valuable Te-based nanomaterials for future technological uses.The present study highlights the capability of the strictly aerobic Rhodococcus aetherivorans BCP1 strain (DSM 44980) was cultured as described elsewhere21, whose details are indicated in the Supplementary Information. The number of viable cells is reported as average of the Colony Forming Unit (log10[CFU mL−1]) for each biological trial (n = 3) with standard deviation (SD). All the reagents were purchased from Sigma-Aldrich®.32− removal by BCP1 resting cells during the exposure timeframe was estimated as published elsewhere52 and described in detail in the Supplementary Information. The data are reported as average (n = 3) of the percentage value corresponding to TeO32− removal over the incubation time with SD. Further, since any statistical difference was observed between the CFU mL−1 counted at the earliest stages of BCP1 resting cells incubation to each oxyanion concentration tested, the specific rate of TeO32− bioconversion (expressed as μg mL−1 h−1) was calculated using a linear regression of the data collected over 3 h.The extent of TeO21, while Transmission Electron Microscopy (TEM) imaging of both TeO32−-exposed BCP1 resting cells and Te-nanostructure extracts was performed using a Hitachi H7650 TEM. For bright field (BF) and high-resolution (HR) TEM, as well as the corresponding Selected-Area Electron Diffraction (SAED) pattern of TeNRs were collected by FEI Tecnai F20 TEM at an acceleration voltage of 200 kV. The samples were prepared by mounting 5 µL of either cellular suspensions or Te-nanostructure extracts on carbon-coated copper grids , which were air-dried prior the imaging. TEM micrographs were analyzed through ImageJ software to measure the actual length of TeNRs, which was calculated considering 100 randomly chosen nanorods. The distribution was fitted to a Gaussian function to yield the average length of TeNRs.Te-nanostructure extracts were recovered from BCP1 resting cells following the procedure published in our previous study21. As a control experiments, a liposome solution (5 mM) was prepared by dissolving in 5 mL of water 19 mg of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) , which is non-ionic surfactant53. The POPC liposome solution and the isolated amphiphilic biomolecules were extruded through a Mini-Extruder equipped with a polycarbonate membrane (0.1 μm) .For experiments aimed to evaluate the presence of amphiphilic biomolecules within the TeNRs extract, the sample containing the amphiphilic biomolecules without biogenic nanomaterial was isolated as described elsewhereDLS measurements were performed onto 1 mL of (i) the isolated and extruded amphiphilic biomolecules and (ii) the POPC liposome solution by using Zen 3600 Zetasizer Nano ZS™ from Malvern Instruments.18(3) Invitrogen™) dissolved in methanol was used as the probe in all experiments. 3 mL of DiOC18(3) stock solution (0.4 mM) was utilized to obtain fluorescence spectra of the free dye. TeNRs extract, as well as the isolated and extruded amphiphilic molecules were incubated with the dye previously dried under argon flow, incubating 3 mL of each sample for 30 minutes at room temperature with shaking. Further, the POPC liposome solution was dried along with the dye under argon flow, then resuspended in 3 mL of water to incorporate the lipophilic tracer within the liposomes. The samples were excited (λex) at 488 nm and fluorescence emission spectra were collected above 495 nm wavelength using a Varian Cary Eclipse fluorescence spectrophotometer, while excitation spectra were obtained for 3 different fixed wavelengths .The lipophilic tracer 3,3′-dioctadecyloxacarbocyanine perchlorate (DiOCex = 488 nm). All the autocorrelation functions were built by the Vistavision ISS software and fitted according to a theoretical model for three-dimensional (3D) global diffusion, assuming that the detection volume was approximated by a 3D Gaussian function54. Based on the fitted autocorrelation functions, for each sample the diffusion coefficient and the time of diffusion were calculated.FCS experiments were carried out with an ISS Alba IV Confocal Spectroscopy & Imaging Workstation coupled with a Nikon Eclipse Ti-U microscope. The lipophilic tracer was diluted to a final concentration of 2 nM, and 400 μl of this dilution was used to perform FCS. The autocorrelation curves corresponding to all the samples were obtained from 15 independent runs by exiting the dye with a single photon CW Ar-laser  to enhance the resistance values recorder by 5492B Digit Multimeter (BK PRECISION®). The obtained resistance values correspond to the average of 6 independent measurements with SD.The electrical property of the TeNRs extract was studied by air drying 800 μL of sample onto a 2 × 1 cm Crystal Silicon wafer , whose contacts were drawn through silver painting .Supplementary Information


The specific amyloid-beta (Aβ) species or other amyloid-precursor protein cleavage products that are most directly related to human neurodegeneration and clinical dementia of the Alzheimer’s type have not yet been directly identified. Without a clear understanding of the most relevant species, it is difficult to determine whether therapeutic candidates successfully engaged the correct target(s). Here, we review some of the controversies regarding soluble Aβ aggregates  and propose experiments designed to move forward towards new therapeutic approaches. Specifically, we review the increasing evidence for the relevance of non-canonical forms of Aβ, the much more potent toxicity attributable to native species than to synthetic Aβ, and the evidence implicating the ratio of soluble Aβ aggregates to plaques in differentiating demented patients from non-demented high Aβ plaque pathology controls. To move forward, we propose four related directions. 1) Narrowing the focus to species derived from human Alzheimer’s disease (AD) brain tissue, as opposed to synthetic Aβ, cell culture-derived species, or species primarily present in animal models. 2) Careful study of differences between patients with dementia of the Alzheimer’s type vs. non-demented controls with high Aβ plaque pathology. This will involve testing the hypothesis that, under some circumstances, plaques may buffer soluble toxic species, but later release them into the surrounding milieu. 3) Investigations of other protein constituents of soluble Aβ aggregates in addition to Aβ itself. Our initial data based on chemical cleavage experiments indicate that other proteins do appear to be part of the human brain soluble Aβ aggregates. 4) Multimodal experimental assessments of toxicity, including longer term effects on synapse loss, related deleterious cellular responses, and degeneration in human-derived neuron-like cells. Overall, the goal is to identify specific Aβ species, other amyloid precursor protein cleavage products, or other key proteins in aggregates present in human AD brains, less abundant in non-demented high pathology control brains, and robustly toxic in a wide variety of relevant assays. These species themselves, the enzymatic or cellular processes involved in their production, and their routes of clearance would be highly relevant therapeutic targets for dementia of the Alzheimer’s type. Genetic linkages in humans using clinical status as the outcome measure represent the highest level of evidence currently available regarding causal relationships in dementia of the Alzheimer’s type. Successful clinical trials would offer a higher level of evidence, but as yet these have not been reported.The Aβ species present in the human brain are the most directly relevant. Synthetic, cell culture-derived, and animal Aβ may not be composed of the most relevant proteoforms and can take a wide variety of aggregation states, not all of which may be present in the human brain.Synapse loss with or without more widespread neurodegeneration is likely to play an important role in clinical dementia. This assertion is based on post-mortem findings in human brain indicating that the extent of synapse loss differentiates patients with dementia of the Alzheimer’s type from high plaque pathology non-demented control subjects . The rolThe amyloid cascade hypothesis has been a mainstay of AD research for decades . The genThe most compelling genetic evidence favoring a key role for Aβ or another related amyloid-precursor protein (APP) fragment can be briefly summarized as follows: mutations that cause increases in amyloid precursor protein (APP) production or alterations in cleavage favoring certain products lead to early onset dementia ; an APP This genetic evidence still leaves considerable room for many alternative explanations. While Aβ plaques are one of the key pathological hallmarks of AD, they are not sufficient to cause dementia of the Alzheimer’s type; many cognitively normal elderly individuals have plaques that are as yet indistinguishable qualitatively and quantitatively from those of demented patients –12. PlaqThe species is an Aβ dimer made of two non-canonical Aβ peptides, e.g., extended, truncated, or post-translationally modified forms. The plausibility of this alternative is supported by the recent findings from Portelius et al. –27, and The species is a non-canonical Aβ peptide stably linked to another molecule that increases its size. Such species have not yet been identified in human brain, but appropriate methods to identify them are lacking.The species is a new amyloid-precursor protein fragment with Aβ mid-domain immunoreactivity but N-terminal and/or C-terminal extensions. Recent evidence for the existence of toxic species in cell culture systems lends plausibility –31, but The species is a canonical Aβ peptide stably linked to another molecule that increases its size. This initially seems unlikely given that the HJ5.1- HJ3.4 ELISA should detect such species if they are present. However, the other molecule could obstruct the binding of either of the antibodies used in ELISA, even under denaturing conditions.Less likely explanations include:The species is a bona fide Aβ dimer made of two canonical Aβ peptides. This seems unlikely given that we and others have not observed a species of this size using sensitive ELISAs that should detect such species. The ELISAs use HJ3.4 and 82E1, antibodies that recognize the free N-terminus of canonical Aβ to both capture and detect Aβ.The species is an Aβ dimer made of one canonical Aβ peptide and one non-canonical Aβ peptide. This seems unlikely given that we and others have not observed a species of this size using a sensitive ELISA that should detect such species. The ELISA uses a mid-domain antibody HJ5.1 to capture and the canonical N-terminus-specific antibody HJ3.4 to detect.The species is an N-terminal canonical but C-terminally extended amyloid-precursor protein fragment. This seems unlikely given that the HJ5.1- HJ3.4 ELISA should detect such species if they were present.Shankar et al.  observed1-40 and Aβ1-42, but more precise methods such as tandem mass spectrometry that can definitively identify the Aβ species were not performed. Again, many possibilities exist for the identities of the amylospheroid structures.Noguchi et al.  reportedLesne et al. reported an approximately 56 kDa species that appears to play a key pathophysiological role in some transgenic mouse models  but instIn our studies, the soluble Aβ aggregates from human AD brain were predominantly very high molecular weight, eluting close to the void volume of a Superdex 200 size exclusion chromatography column (>670 kDa based on globular protein standards) , 24. MasThus, it appears likely that several discrete types of soluble Aβ aggregates are present in the human brain which may exert different types of toxicity. However, none of the previously reported soluble Aβ aggregate species were assessed explicitly in high plaque pathology non-demented controls, so it is unclear if their concentrations correlate with Aβ plaques or correlate with dementia of the Alzheimer’s type. Furthermore, none of these species have been sufficiently purified to allow accurate structural characterization. Thus, apart from proprietary conformational-specific antibodies, there are no generalizable leads for constructing pharmacodynamic assays or developing targeted therapeutics.We reported in 2013 that the concentration of soluble Aβ aggregates from patients with dementia was higher than the concentration of soluble Aβ aggregates from control subjects with indistinguishable plaque burden but no dementia. The ratio of soluble Aβ aggregate concentration to plaque area fully distinguished these two groups of subjects, with no overlap between groups  + plaque brains vs. those isolated from CDR1 (demented) + plaque brains on frozen (not fixed) slices from CDR0 + plaque vs. CDR1 + plaque brains. This would allow testing of three specific sub-hypotheses:We propose the following line of investigation to test the plaque buffering hypothesis:and qualitative changes in soluble aggregates, and quantitative changes in soluble aggregates could correlate with dementia. This would be difficult to fully interpret. A null result is also possible: plaques do not buffer soluble Aβ aggregates. This could indicate a limitation of the methods used or indicate that plaque buffering does not play a major role.It is possible that there could be a mixed result: qualitative changes in plaques, The strong buffering presumes that the endogenous soluble Aβ aggregates associated with the slices have been largely washed out during the preparation of the slices for binding studies. Recent unpublished results from Dominic Walsh’s group indicate that at least some species of endogenous soluble Aβ aggregates do in fact diffuse out of minced tissue . The readily diffusible species may represent only a subset of the soluble Aβ aggregates extracted during tissue homogenization.A technical hurdle will involve labeling the isolated endogenous aggregates without interfering with their binding characteristics. Biotinylation, tritiation, and iodination of N-terminal residues, C-terminal residues, or tyrosine residues could be considered. Competition with unlabeled endogenous aggregates would be an appropriate control to assess the relative binding affinity of labeled vs. unlabeled aggregates. If the labeling protocol alters the characteristics of the endogenous soluble Aβ aggregates, the affinity of binding vs. the affinity of competition would be different. If the affinity of binding and the affinity of competition are found to be similar, this would indicate that the labeling does not markedly alter the endogenous soluble Aβ aggregates. At the same time that plaque buffering capacity is being tested, the endogenous labeled aggregates could also be tested for labeling to other structures such as synapses, microglia, astrocytes, and blood vessels. This could either confirm or refute the relevance of the observation that synthetic Aβ oligomers bind specifically to synapses .The results of these experiments will direct the critical next steps: If qualitative changes in the soluble Aβ aggregates correlate with dementia, an extensive search for the structural differences between soluble Aβ aggregates from controls without dementia vs. soluble Aβ aggregates from patients with dementia would be well justified. Biochemical purification and mass-spectrometric characterization could be appropriate methods. Top-down (undigested) proteomic methods could be used to identify and quantify the specific proteoforms of Aβ in the aggregates from demented vs. non-demented controls . The enzWe envision a scenario in which Aβ production and accumulation are left to proceed , but an enzyme responsible for conversion of a less toxic form of soluble Aβ to a more toxic form is inhibited. The patients, hopefully, would live long lives with brains full of benign plaques but no dementia of the Alzheimer’s type.1-42 peptides includes only 16 out of the 20 common amino acids . It could be, of course, that these are impurities that are nonspecifically co-immunoprecipitated. It is also possible that they are true components of the high molecular weight soluble aggregates, which would then be best described as hetero-oligomeric or multicomponent aggregates. This is not an easy question to address using biochemical purification methods alone; the conditions required to obtain very high specificity during the immunoprecipitation may also break up true non-covalent aggregates. An orthogonal approach, upon which we have begun to embark, is to degrade other proteins in a fashion which does not affect Aβ and then ask what effects this has on the size forms of soluble Aβ aggregates from human AD brain Fig. . If the ids Fig. . However1-40S26C containing cysteine in place of serine 26 to produce a disulfide cross-linked dimer. Most importantly, though, these results indicate that DTT and NTCB together have no effect on the soluble Aβ aggregate assay within the range of the high molecular weight Aβ aggregates in fractions 7 to 10. Thus, these chemicals caused some artifacts in the very low molecular mass region, but not in the higher molecular weight regions where soluble Aβ aggregates would be expected to be found after size exclusion chromatography  would be feasible. Control proteins containing at least one cysteine were readily cleaved, whereas synthetic Aβ was not Fig. . To testphy Fig. .In advance, we specified three possible non-mutually exclusive quaternary structural hypotheses Fig. . In the Our proposal for moving forward involves assessing the characteristics of the most toxic species present in human brain from patients with dementia of the Alzheimer’s type. The species should correlate with dementia, i.e., they should not be present or occur at lower concentrations in otherwise similar non-demented controls with Aβ plaque pathology. The characterization of the species could start with sensitive immunoassays based on a variety of antibodies to assess the N-terminal extended region, canonical N-terminus, mid-domain, canonical C-terminus, and C-terminal extended regions. We should not assume that they consist of canonical Aβ; many other non-canonical species with substantial aggregation potential and toxicity have been reported , 50–52. There may be more than one major class of toxic soluble Aβ aggregates. Liu et al.  proposedA critical question in the field is which in vitro or in vivo model system best recapitulates the key aspects of neurodegeneration underlying dementia of the Alzheimer’s type. The mainstay of investigations into neurodegenerative effects have involved assessments of cell and synapse loss in rodent neuronal cell culture systems, effects on synaptic plasticity and structure in rodent brain slices, and behavioral and morphological assessments in animal models. None of the typical toxicity assays involve human cells. De Strooper’s group recently demonstrated that human induced pluripotent stem cell (IPSC)-derived neurons were more susceptible to degeneration than mouse IPSC-derived neurons when transplanted into the brains of APP transgenic immunodeficient mice . This syOnce the characteristics of the most toxic species present in human AD brain have been determined, quantitative methods for tracking the species during purification should be employed; an ELISA or similar relatively high throughput assay would be most appropriate. Quantitative bookkeeping should be used to ensure that the purified species represent the predominant species, rather than a small minority species that is selectively enriched. Then, after purification, high resolution tandem mass spectrometry could be used to specifically identify non-canonical forms of Aβ , N-termiLooking forward into the future, if one or more human brain toxic species can be specifically characterized, a critical next step will be to develop pharmacodynamic assays for use in human patients. With good pharmacodynamic assays, quantitative assessment of target engagement in early phase clinical trials can be performed to guide dosing in human patients. Without good pharmacodynamic assays, it will be impossible to tell whether candidate therapeutics actually cross the blood–brain barrier and engage the target. In our view, none of the failed clinical trials in AD have compellingly demonstrated target engagement, irrespective of the question of whether they were targeting the right target. Thus, while some have stated that the amyloid hypothesis is on its last legs, it may be more correct to state that it has not yet been sufficiently tested.


We observed that the upper thermal limits of leaf function in 12 tree species from a seasonally dry tropical forest were perilously close to maximum temperatures in this region. Exposure to drought increased thermotolerance, and species that were more drought tolerant were also more thermotolerant. Importantly, thermotolerance was positively related to the key leaf functional trait - leaf mass per area, and negatively related to photosynthetic rates. These results indicate that tropical trees will be vulnerable to increased warming, and more productive species with lower LMA and higher photosynthetic rates may be more negatively affected by global warming and future climate change. 50) in experimentally grown saplings of 12 tree species from a seasonally dry tropical forest. We examined the relationship of thermotolerance with leaf functional traits and photosynthetic rates. Additionally, we tested how water limitation altered thermotolerance within species, and examined the relationship between thermotolerance and drought tolerance among species. Thermotolerance ranged from 44.5 to 48.1 °C in the least and most thermotolerant species, respectively. The observed variation in thermotolerance indicates that the upper limits of leaf function are critically close to maximum temperatures in this region, and that these species will be vulnerable to, and differentially affected by, future warming. Drought increased temperature tolerance, and species that were more drought tolerant were also more thermotolerant. Importantly, thermotolerance was positively related to the key leaf functional trait—leaf mass per area (LMA), and congruent with this, negatively related to photosynthetic rates. These results indicate that more productive species with lower LMA and higher photosynthetic rates may be more vulnerable to heat and drought stress, and more likely to be negatively affected by future increases in extreme climatic events.Understanding how tropical trees will respond to extreme temperatures and drought is essential to predict how future increases in the severity, frequency and duration of extreme climatic events will affect tropical systems. In this study, we investigated leaf thermotolerance by quantifying the temperatures that resulted in a 50 % decrease in photosystem II function (T With future global warming, plants will experience extreme temperatures more frequently , and estimated the temperature that results in a 50 % loss of function (T50). The temperature response of chlorophyll a fluorescence is a physiological measure of the integrity of the thylakoid membrane, is highly sensitive to high temperature, represents a good indicator of photosynthetic and organismal thermotolerance . This allowed us to identify and select representative species that span the range of leafing behaviour and leaf functional trait values observed in this region see .We selected 12 tree species that are commonly found in the seasonally dry forests of the Northern Western Ghats of peninsular India see Supporting Information—Table S2 for details of height and stem diameter] were obtained from a local nursery , and transplanted to 19 L PVC pots  filled with 18 kg dry red alfisol (pH 7.2) supplemented with organic manure (1:50 v/v). Saplings were moved to a greenhouse and given a period of 6 weeks to acclimate before the experiment. All plants were fertilized once, 15 days after transplantation, with urea (0.05 g·kg−1 soil). The greenhouse received natural sunlight supplemented with incandescent lamps to ensure photosynthetic photon flux density (PPFD) of 500–800 µmol·m−2·s−1 (between 0900 and 1600 h). Mean daily temperature in the greenhouse ranged between 25 and 31 °C, while relative humidity (30–55 %) varied according to local conditions.The study was conducted between May and July 2015 at the Indian Institute of Science Education and Research (IISER) campus, Pune, India. For all species, 12 individuals of 2.5-year-old saplings [At the start of the experiment, the 12 individuals of each species were randomly assigned to the two treatments  and randomly allocated positions in the greenhouse. Before the beginning of the treatments, all pots were fully saturated with water in the evening, excess water allowed to drain overnight and weighed the next morning to determine the pot weight at field capacity. A white plastic sheet was taped to the pot rim and loosely tied around the base of the plants to minimize evaporation from the soil. During the experiment all control pots were individually weighed every 3 days, the loss of water quantified and the appropriate volume of water added to bring the pot back to 90 % of its field capacity. In this manner all control plants were always maintained at 75–90 % field capacity.Garcinia indica because of the lack of sufficient plants. At the end of the drought treatment we measured gas exchange, quantified leaf wilting and collected leaf samples for thermotolerance assays and estimation of leaf RWC.Drought was imposed by termination of watering at the start of the experiment. These pots were weighed every 3 days to estimate water loss and determine the point at which the pot water reached 30 % of field capacity. To standardize the drought treatment across these species that varied widely in their water use, we used the time at which pots reached 30 % of field capacity as the end point of the drought treatment. This pot water content was chosen to ensure that all species showed significant signs of water limitation, but also so that the drought treatment did not result in severe leaf necrosis and death in any of the species. The affect of drought was not examined for Average leaf wilting stage scores were estimated and RWC quantified in the morning after drought-stressed plants reached 30 % of field capacity. Average leaf wilting was scored in a semi-quantitative manner (2) were excised with a cork borer from the middle of the first fully expanded mature leaf taking care to exclude the midvein. The discs were weighed to quantify fresh weight (FW), water saturated for 24 h at 4 °C and subsequently the turgid fresh weight (TW) measured. Leaf discs were then put in a drying oven at 70 °C for 3–4 days till a constant dry weight (DW) was obtained. Leaf RWC  fitted with the LICOR-6400-02B LED light source. These measurements were made between 0930 and 1130 h with the cuvette light, CO2 concentrations (incoming reference), relative humidity and temperature set at 800 μmol m2 s−1 PPFD, 390 ± 10 ppm, 50–60 % and 28–30 °C, respectively.Leaf gas exchange was measured for the first fully expanded leaf for six individuals each for control (well watered) and drought-stressed plants (at the end of the drought treatment) with a LI-6400XT portable photosynthesis system  using the standard broadleaf cuvette (6 cm−2), LDMC (mg g−1) and leaf area  were quantified for five fully expanded and mature leaves from six individuals of the control (well watered) plants as per protocols recommended by LMA (g ma fluorescence (Fv     /Fm), and estimated the temperature that results in the 50 % loss of function (T50). Fv   /Fm represents the maximum potential quantum yield of PSII and was calculated as Fv   /Fm = (Fm − Fo)/Fm, where Fm and Fo are the maximum and basal fluorescence yield, respectively, for dark-adapted leaves. Leaf discs (2 cm2) were placed between two layers of muslin cloth, covered with aluminium foil and put in a sealed plastic lock bag. A moistened wad of tissue paper was put in the bag to maintain high water vapour content and prevent dehydration in the leaf. The plastic bag was immersed in a temperature-controlled refrigerated water bath  set to achieve the desired leaf temperature  for 30 min. Separate leaf discs from independent leaves from the same individuals were used for each temperature. Preliminary trials and previous studies .For control (well watered) plants, we measured the temperature response of dark-adapted chlorophyll a fluorescence (Fv   /Fm) values across the range of temperatures examined using the R package ‘drc’ (50) at which reduction in Fv      /Fm was 50 % of the upper asymptote was estimated from these curves. We used five independent leaf discs from an individual at each of the temperatures to generate an Fv     /Fm response curve from which we estimated T50 for that individual. This was repeated for 5–6 individuals for each species. For the drought-stressed plants we measured Fv   /Fm at 25, 47.5, 50 °C due to limited availability of leaf samples, and thus did not calculate T50. Based on knowledge that variation between species is maximal at 47.5 °C, we use PSII function at this temperature as an index of thermotolerance that reflects the relative ranking of species performance at high temperatures.A four-parameter logistic sigmoid curve was fitted to the chlorophyll ge ‘drc’ . The fou50 of PSII function for the control plants using a one-way ANOVA with species as a fixed effect. To test how experimentally imposed drought affects thermotolerance in these species, we examined variation in Fv   /Fm using an ANOVA with species, treatment (control and drought) and temperature  as fixed effects. Variation in LMA, LDMC, LA, wilting score and RWC was examined using ANOVA with species as a fixed effect. We performed non-parametric Kruskal–Wallis tests for LMA, LA and wilting scores as these were not normally distributed. Variation in photosynthetic rates was examined with a two-way ANOVA with species and treatment (control and drought) as fixed effects.We examined variation in T50 of PSII for control, and Fv   /Fm at 47.5 °C for drought treatments), leaf traits  and drought tolerance . Additionally, we examined Spearman’s rank correlations for analyses with LMA, LA and wilting scores as these variables were not normally distributed. All analyses were performed using Statistica .We used Pearson’s correlations to examine relationships between thermotolerance (TFv   /Fm ranged from around 0.7 to 0.8 at control temperature (25 °C) in all species, and this remained unchanged till temperatures increased above 40 °C .Dark-adapted ve 40 °C . There wbellatum . While nFv   /Fm for plants that were drought stressed see . As with plants that were well watered, Fv   /Fm decreased significantly at 47.5 °C, and was very low at 50 °C . However, drought-stressed plants performed significantly better than well-watered plants at these temperatures. The highest variation in Fv   /Fm between species was observed at 47.5 °C, and we used Fv   /Fm at 47.5 °C as a relative index of thermotolerance for the drought-stressed plants to test for differences between species, the effect of drought and the interactions between the two. Species differed significantly from each other, and drought resulted in better performance at this stressful temperature .At control temperatures (25 °C) there was no difference in dark-adapted perature . The incsee . LMA and LA were not normally distributed and Kruskal–Wallis non-parametric analysis yielded similar results with significant differences between species for both traits. Photosynthetic rates varied nearly 2-fold from the least productive species to the most productive species under well-watered conditions species see . Photosynthetic rates decreased sharply in drought-stressed plants, but the magnitude of decrease differed among species see . Average leaf wilting scores were significantly different across species and ranged from one in M. umbellatum indicating no wilting, to greater than three in Bridelia retusa indicating severe wilting and the beginning of leaf necrosis see . Leaf wilting scores were not normally distributed and Kruskal–Wallis non-parametric analysis yielded similar results with significant differences between species. Similarly, there was significant variation in leaf RWC which ranged from around 30 % to greater than 80 % see .The leaf traits measured, LMA, LDMC and LA differed significantly among species 50 of Fv   /Fm, and Fv   /Fm at 47.5 °C, respectively, was positively related to LMA, but not to LDMC or leaf size (50 (control plants), but not for Fv   /Fm at 47.5 °C (drought-stressed plants). Spearman’s rank correlations were not significant for either T50 of Fv   /Fm in control plants, or Fv   /Fm at 47.5 °C for the drought-stressed plants.Thermotolerance for both control and drought-stressed plants as measured by Teaf size . As LMA 50 of Fv   /Fm in control plants, and Fv   /Fm at 47.5 °C drought-stressed plants) was negatively related to photosynthetic rates for control plants, but not related to photosynthesis in the drought-stressed plants (P < 0.1).Thermotolerance , which represent temperatures that cause irreversible damage and necrosis . Future increases of 6 °C in maximum temperatures would exceed the T50 for all but the most tolerant species.Future global warming and increased frequency of exposure to higher temperatures will likely have severe negative consequences for trees in this region. Temperatures in the tropics are predicted to increase by 3–6 °C over the course of this century , and forThe observed relationship of thermotolerance with LMA and photosynthetic rates implies that species vulnerability to future climate change may not be random but related to functional attributes. This is particularly insightful because this allows us to extrapolate our results to other species beyond those examined in this study. Our results show that more productive species with lower LMA and higher photosynthesis are less thermotolerant, and therefore likely to be more vulnerable to future global warming, and this may result in directional changes in future species composition favouring species with high LMA and lower photosynthesis.The LMA–thermotolerance relationship is congruent with predictions made by We observed higher average thermotolerance in evergreen than in deciduous species, as previously reported in tree species from this region . Leaves Water limitation and drought resulted in the ability to withstand higher temperatures in these species that are adapted to hot and dry conditions. This has been documented in other plants , India.D.B., A.G. and A.S. conceived the study, designed the experiments, collected and analysed the data, and drafted the manuscript.None declared.The following additional information is available in the online version of this article—Table S1. List of study species, family names, leaf habit and species codes used.Table S2. Height and stem diameter of plants at the beginning of the experiment.Table S3. Variation in thermotolerance between evergreen and deciduous leaf habit.Table S4. Variation in thermotolerance  for 11 species under control (well watered) and drought-stressed conditions.Table S5. Variation in leaf functional traits for the 12 species: a) leaf mass per area (LMA); b) leaf dry matter content (LDMC); and c) leaf size (LA).Table S6. Variation in photosynthetic rates for the 11 species under control (well watered) and drought conditions.Table S7. Variation in drought performance in 11 species as evaluated by: a) leaf wilting stage scores; and b) leaf relative water content (RWC).Figure S1. Climate data for the region from .Figure S2. Canopy fullness for study species from Bhimashankar Wildlife Sanctuary in the Northern Western Ghats, India.Figure S3. The effect of drought stress on dark-adapted chlorophyll a fluorescence (Fv/Fm) at 25, 47.5 and 50 ŶC.Figure S4. Leaf functional traits in the 12 study species: a) leaf mass per area; b) leaf dry matter content; c) leaf area.Figure S5. Performance of the 12 species: a) photosynthesis under control and drought conditions; b) leaf wilting stage scores under drought conditions; c) leaf relative water content under drought conditions.Supporting InformationClick here for additional data file.


Flash sintering has attracted significant attention as its remarkably rapid densification process at low sintering furnace temperature leads to the retention of fine grains and enhanced dielectric properties. However, high-temperature mechanical behaviors of flash-sintered ceramics remain poorly understood. Here, we present high-temperature (up to 600 °C) in situ compression studies on flash-sintered yttria-stabilized zirconia (YSZ). Below 400 °C, the YSZ exhibits high ultimate compressive strength exceeding 3.5 GPa and high inelastic strain (~8%) due primarily to phase transformation toughening. At higher temperatures, crack nucleation and propagation are significantly retarded, and prominent plasticity arises mainly from dislocation activity. The high dislocation density induced in flash-sintered ceramics may have general implications for improving the plasticity of sintered ceramic materials. Flash sintering allows for rapid ceramic processing, but the mechanical behavior of such ceramics remains poorly understood. Here, the authors compress micropillars of yttria stabilized zirconia to show flash sintering promotes outstanding plasticity. One promising candidate for thermal barrier coatings is yttria-stabilized zirconia (YSZ). YSZ offers one of the lowest thermal conductivities, ~2.3 W/mK at ~1000 °C2. Monolithic zirconia has monoclinic phase (space group P21/c) at room temperature. However, the tetragonal phase (space group P42/nmc) of zirconia can be stabilized by doping zirconia with Y2O3, CeO2, and MgO4. The discovery of martensitic phase transformation  in ZrO2 has led to significant investigations on its deformability. The volume expansion (~4%) during martensitic phase transformations near the crack tips induced by an external stress can introduce compressive stress that can in turn retard crack propagation6. Therefore, YSZ provides new opportunities for various applications, including reliable thermal and environmental barrier coatings, solid oxide fuel cells, and shape memory devices, just to name a few8.Ceramic materials have a variety of high-temperature applications, such as thermal barrier coatings for high-thrust engines and turbines9. Certain nanostructured ceramics have shown high strength, wear resistance, and/or fracture toughness at elevated temperatures12. However, conventional sintering typically requires very high temperature and long sintering time and thus leads to significant grain coarsening13. Recently, it has been discovered that YSZ can be fully densified within a few seconds at a temperature much lower than conventional sintering temperature by a novel sintering technique named flash sintering14. The ultrafast sintering technique enables the retention of nanograins and enhanced dielectric properties15. Flash sintering occurs by applying a ramp heating process at a constant heating rate under moderate electrical fields24. Once the temperature is above the onset of the flash temperature, under applied electrical field, a densification process takes place within a few seconds as evidenced by a sudden increase of electrical conductivity, accompanied with drastic increase in mass density25.A majority of ceramic materials possess high strength but low toughness at low temperatures due to the lack of dislocation-enabled deformability30. However, our understanding of the mechanical behaviors of small-scale YSZ specimens at elevated temperatures remains limited. Recently, Korte and Clegg showed that microcompression tests on small specimens could be carried out at an elevated temperature (~500 °C) by heating the sample stage and indenter tip without a significant mechanical and thermal drift (~1 nm/s)32. High-temperature micropillar compression technique enables the study on the temperature-dependent deformation mechanisms for brittle materials at elevated temperatures. Furthermore, the deformability of flash-sintered ceramics is largely unknown despite their intriguing microstructures, including the generation of a large number of charged defects during the flash-sintering process25.Prior studies on the mechanical behavior of YSZ showed superelasticity and shape memory effect at room temperatureHere, we report an in situ micropillar compression study on the deformability of a flash-sintered 3 mol% yttria stabilized zirconia (3YSZ) at elevated temperatures (up to 600 °C). The flash-sintered 3YSZ contains abundant dislocations. The mechanical behaviors of the flash-sintered 3YSZ are highlighted by increased plasticity, and temperature-dependent transition of deformation mechanisms.33. The applied heat and electrical field were removed right after the onset of flash sintering to prevent grain growth. Even though the densification process of 3YSZ occurred at relatively low temperature, X-ray diffraction pattern shows a dominant tetragonal phase without evident monoclinic and cubic phases  was heated in a thermomechanical analyzer with platinum electrodes at a constant heating rate of 25 °C/min under an electrical field of 150 V/cm. The ultrafast densification process occurred at a furnace temperature of 1150–1200 °C in a few seconds . Figure Micropillars with ~3 μm in diameter and ~6 μm in height were fabricated using the focused ion beam (FIB) technique on the flash-sintered 3YSZ. Given that the average subgrain size of the specimen is ~160 nm, each pillar contains more than 5000 subgrains. Thus, the mechanical behaviors of the micropillars are a good representation of a large number of grains. Uniaxial in situ microcompression tests on the micropillars were carried out from room temperature to 600 °C at a constant strain rate of 5 × 10Figure Meanwhile, we carried out TEM experiments on the YSZ pillars after microcompression tests tested at room temperature. Supplementary Fig. −3 s−1 at room temperature and 400 °C as shown in Fig. Thirty cyclic loading and partial unloading tests were carried out at a strain rate of 5 × 1025. This assertion coincides with TEM studies of 3YSZ sintered under 1.5 and 15 V/cm, which display insignificant grain growth and little dislocations. Rapid grain growth and large electric field may play a significant role in producing dislocations and their arrays by generating the internal stress during flash sintering. The electric field of 1.5 and 15 V/cm is insufficient to induce dislocations in 3YSZ. Interestingly, intragranular dislocations and dislocation entanglement in 3YSZ have been observed at an ultrahigh temperature, 1400 °C, during tensile creep tests at 50 MPa36. In the current study, the generation of intragranular dislocations, their arrays and entanglement near triple junctions may strongly depend on stress and high strain rate plastic flow during flash sintering. The shear stresses allowing dislocation pile-up in 3YSZ range from 350 to 1260 MPa based on the lattice dislocation pile-up model37:In general, dislocations are rare in ceramics as the strong covalent and ionic bond greatly discourages the formation of dislocations. However, TEM studies herein show ample evidence for the formation of high-density dislocations in flash-sintered 3YSZ. Also, dislocation arrays are frequently observed near triple junctions. It is likely that a large stress concentration (gradient) develops near triple junctions during the rapid grain growth process. Significant mass (atomic) transport occurs during the flash-sintering process to fill in the voids among grains/particles during a very short sintering time . The high rate  of mass transport and flow near triple junctions may lead to substantial plastic deformation , which is within the range that can form dislocation arrays in 3YSZ at elevated temperatures during flash sintering. The dislocation density in many grains reaches as high as ~2–3 × 1012 m−2, comparing to the typical density of ~108–1010 m−2 in a majority of ceramics38. These high-density dislocations play a vital role on the deformability of the flash-sintered 3YSZ tested at elevated temperatures. It is well known that dislocation glide is unlikely to take place in bulk ceramic materials at room temperature unless applying a confinement pressure via hydrostatic or gaseous medium31. However, at microscales, plastic deformation of certain ceramics by glide of dislocations has been observed40. Camposilvan et al. investigated the plasticity of conventionally sintered YSZ at small scales at room temperature and speculated that dislocation activity along with transformation-induced plasticity at a higher stress level (without showing direct evidence of dislocations) may be a possible inelastic deformation mechanism for YSZ28. Our finite-element method analysis on stress distribution for dislocations in polycrystalline YSZ further supports this assertion and shows that shear stress concentration near-grain boundaries and triple junctions induces the nucleation and migration of dislocations, thereby facilitating the plastic deformation of YSZ . Thus, the large ~8% inelastic flow may arise from not only transformation-induced plasticity, but also dislocation activity especially at higher stress level.A strain of ~8% for stabilized zirconia owing to martensitic transformation-induced plasticity has been previously reported37. However, at 400 °C, cracks initiated from the top surface of the micropillar propagate downward gradually and slowly without catastrophic failure unlike the brittle catastrophic fracture of micropillars tested at room temperature. The prominent variation of fracture morphology of the deformed pillars implies that a new inelastic deformation mechanism supersedes martensitic transformation toughening as temperature rises. In the conventionally sintered bulk YSZ system, it is known that the 700–800 °C temperature range favors other mechanisms  as a substitute to martensitic transformation41. However, the flash-sintered YSZ contains nanograins, oxygen vacancies15, and abundant preexisting dislocations, which may result in the early activation of other inelastic deformation mechanisms at 400 °C42. It is worth mentioning that the critical strain for the pillars compressed at 200 °C is still high (~6%). Catastrophic failure of pillars was also observed for specimens tested at 200 °C, and transformation toughening-induced plasticity remains the dominant inelastic deformation mechanism of the flash-sintered 3YSZ.For micropillars tested at 400 °C, cracks nucleated at smaller strain, ~4%, due to the lack of martensitic transformation toughening. At elevated temperatures, the metastable tetragonal phase begins to thermally transform into a mixture of tetragonal and cubic phase, degrading the deformability of the flash-sintered 3YSZ by transformation toughening43.At an even greater test temperature, the martensitic phase transformation toughening is gradually replaced by the activation of a new inelastic deformation mechanism. Basically, the critical strain at which the first crack occurs decreases with increasing test temperature and reaches a minimum at ~400 °C as can be seen in Fig. 28. Taking the underestimation into consideration, it is still surprising that the apparent elastic modulus of the flash-sintered 3YSZ increases with temperature and reaches a maximum at 400 °C  Ceramic materials retaining superelasticity usually exhibit lower elastic modulus due to the strain burst. It follows that the larger elastic modulus observed at elevated temperatures is an indication of suppression of the martensitic phase transformation due to thermodynamically reinforced stability of tetragonal phase48. The total free energy change for martensitic transformation per unit volume (ΔU0) can be expressed as47,The apparent elastic moduli of tested YSZ are lower than the theoretical values 210 GPa) at all test temperatures. It is well known that the underestimation of elastic modulus in the microcompression tests can be attributed to taper angle of the pillars, misalignment between the tips and pillars, and stress concentration on the top surface of the pillars °C Fig. . This is10 GPa atUc is the chemical free energy change, ΔUe the elastic energy change associated with volume expansion of tetragonal zirconia constraint by matrix, ΔUs the interface energy change (negligible), and ΔUI the change in free energy associated with an external applied stress. This mechanical term should be larger than the first three terms on the right-hand side for martensitic transformation to occur. When the chemical free energy term  weakens the interatomic bonds and thus reduces the elastic modulus, and high-density flash-sintered dislocations significantly contribute to the plasticity of 3YSZ as manifested by the increasing critical strain to nearly 7.5% before the observation of cracks at 600 °C. In summary, elastic modulus, critical strain, and fracture behavior of flash-sintered 3YSZ at each test temperature indicate that 400 °C is the temperature beyond which inelastic deformation mechanism of the flash-sintered YSZ changes prominently. Therefore, Zone 1 in Fig. The higher temperature  is observed at both room temperature and 400 °C  is observed at 400 °C and the stress relaxation increases as the pillar undergoes plastic flow. Stress relaxation at high temperature is normally due to high temperature and stress-induced sintering and closure of pores51. However, this mechanism is less likely at the low temperature (400 °C). Thus, stress relaxation taking place at 400 °C may be mainly contributed by a thermally activated inelastic process such as the effect of grain boundary sliding, and/or diffusional creep of dislocations generated during the ultrafast sintering process. Another possible explanation is transient dislocation movement during the holding segment triggered by dislocation gliding, which is more likely to happen at elevated temperatures28. Thus, the observation of stress relaxation may indicates that the dislocation activity contributes to deformability of flash-sintered 3YSZ together with grain boundary sliding and/or diffusion-induced inelastic mechanism above 400 °C. The proposed relaxation mechanisms are all thermally activated phenomenon and time is an important variable. To better understand the inelastic mechanism activated at elevated temperature, systematic strain rate jump microcompression tests are needed in future studies.An indirect way to confirm martensitic transformation is to check the existence of reverse transformationIn conclusion, the first in situ study on mechanical behavior of flash-sintered YSZ was performed inside SEM at elevated temperatures up to 600 °C. At room temperature, YSZ micropillars sustain giant strain (~8%) comparing with its bulk counterpart (~2%) due to the stress-induced martensitic transformation toughening. However, the pillars fracture catastrophically after the nucleation of cracks. In comparison, a brittle-to-ductile transition of fracture mode is observed at 400 °C in flash-sintered YSZ, much lower than the ~800 °C reported in conventional bulk YSZ. The enhanced plasticity at elevated temperatures arises from the transition from phase transformation toughening to dislocation creep, as the dominant inelastic deformation mechanism due to the existence of a high density of dislocations in flash-sintered YSZ and/or to early initiation of grain boundary sliding of ultra-fine grains. This study provides the first evidence on superior mechanical properties of flash-sintered ceramics by using in situ nanomechanical testing technique at elevated temperatures and establishes an important way to fundamentally understand the densification and mechanical properties of ceramics.Flash sintering was performed on a custom-modified thermomechanical testing system . Specimens with a diameter of 5 mm and a thickness of 2 mm prepared by using commercially available 3YSZ  were sandwiched between two platinum electrodes. An alumina rod was utilized to apply minimum pressure (a few kPa) to ensure rigid contact between the electrodes and sample. A DC power of various voltage was applied to achieve electric field of 1.5, 15, and 150 V/cm with a constant heating rate of 25 °C/min (maximum temperature was set to 1300 °C). The experiment was performed in the presence of air. After the onset of flash, the system was switched from the voltage-control mode to a current-control mode. The experiment was terminated right after switching to current-control mode to prohibit grain growth. The linear shrinkage of the samples was measured by a dilatometer.Plan-view TEM samples of flash-sintered 3YSZ were prepared through the conventional approach, which includes manual grinding, polishing, dimpling, and final polishing in an ion milling system . Low energy ion polishing (2 kV) was used to minimize ion milling-induced damage. An FEI Talos 200X TEM/STEM with ChemiSTEM technology (X-FEG and SuperX EDS with four silicon drift detectors) operated at 200 kV was used in this study for microstructure characterization and energy-dispersive X-ray spectroscopy (EDS) chemical mapping.Micropillars of flash-sintered 3YSZ with ~3 µm in diameter and a diameter-to-height aspect ratio of 1:3–1:2 were prepared using focused ion beam (FEI quanta 3D FEG) and a series of concentric annular milling and polishing with progressively de-escalated currents were adopted to reduce tapering angle. Micropillar compression experiments have been performed using a Hysitron PI 88×R PicoIndenter equipped with a piezoelectric actuator on the capacitive transducer that enables the collection of force–displacement data inside a scanning electron microscope (FEI quanta 3D FEG). Moreover, a 20 µm diamond flat punch tip designed for high-temperature compression experiments was used to conduct in situ compression experiments and the geometric variation of micropillars was synchronized to evolving force–displacement curve. For high-temperature in situ compression setups, the flat punch was fastened to probe heater and the specimens were clamped by a V-shaped molybdenum to a ceramic heating stage. The temperature on two heating terminals were simultaneously ramped up at a rate of 10 ˚C/min and isothermally preserved for 3 min before implementing every single compression experiment to eliminate the thermal-driven drifts on both probe and stage sides. An average drift rate of 0.2–0.5 nm/s was estimated in preloading process for 45 s and the estimated force noise level is less than 8 µN prior to compression. An overestimation of specimen displacement during the compression test induced by a displacement associated with the measuring instrument (machine compliance) was systematically measured during in situ SEM studies and corrected.α, β, γ) =  and for the top-left grain,  = . Euler angles operations are: first rotate angle β degrees about the z-axis, then rotate α degrees about the y-axis, and finally rotate γ degrees about new z-axis. The mechanical equilibrium state of the polycrystalline was solved using OOF252, an open source implementation of the finite-element method. The relative numerical tolerance was set to 1 × 10−10. The simulation used 40 GB of RAM and a wall time of ~4 h (See Supplementary Methods for detailed methodology).The microstructure shown in Supplementary Fig. The data that support the findings of this study are available from the corresponding author on reasonable request.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3