The decrease in the ratio of torques evoked by 20 and 100 Hz electrical stimulation, representing low-frequency fatigue, was more evident after the 3 × 30 and 3 × 60 s than the 6 × 5 s bout in both groups and was larger in young than in older women (p < 0.05). Both groups preferred 6 × 5 s cycling for further training. In conclusion, in young women, very low volume (6 × 5 s) all-out exercise induces significant physiological stress and seems to be an effective means of training. For older women, longer exercise sessions (3 × 60 s) are more stressful than shorter ones but are still tolerable psychologically.We investigated the acute neuromuscular and stress responses to three different high-intensity interval training sessions in young (age 19.5 ± 1.3 years) and older (age 65.7 ± 2.8 years) women. Cycling exercise comprised either 6 × 5 s or 3 × 30 s all-out, or 3 × 60 s submaximal, efforts each performed 5 weeks apart in randomized order. Peak and average power was higher in young than in older women and was largest during the 6 × 5 s strategy in both groups ( Reduced physical activity in older adults is associated with increased risk of disease and mortality , 2, and 2+]i), which triggers the mechanism responsible for adaptations. These findings favor “higher-the-better” strategy when considering exercise intensity. All-out efforts of HIIT currently prevails in increasing endurance of various age and physical condition groups i and/or decreased myofibrillar Ca2+ sensitivity [2+]i relationship. Changes in Ca2+ kinetics occur because acute metabolic stress in muscle fibers is accompanied by an increase in the release of reactive oxygen/nitrogen species [Enhanced ion handling during the initial phases of adaptation has been proposed as the main mechanism for improved endurance after exercise training . In the sitivity . This wo species –33 which species . Here, wThe differences in LFF between the young and older women in response to these types of very low volume, high-intensity exercise may be explained by different levels of metabolic stress. Older women were unable to attain high power production during the 5 s bouts to the same level as did young women, and this was reflected in the lower lactate concentration and lower HR percentage relative to maximal HR in the older women. The lower exercise-induced stress may preclude full-scale adaptations, which may require a longer total duration of HIIT, particularly in older women.α concentrations did not change in either group after the 6 × 5 s all-out cycling; this lack of response suggests that impaired Ca2+ handling may be the main mechanism responsible for the effects of extremely low volume, high-intensity training. Such mechanism could be activated only if the metabolic demands are large enough to induce long-lasting LFF.We note that MVC, P100, and IGF-1 and TNF-The findings of our study confirm the strategy of “more is better.” High-intensity cycling for a longer duration induced greater metabolic stress in both young and older women. It was not surprising that the longer exercise was rated as less enjoyable and less preferable for long-term training. This is consistent with previous studies that have compared different exercise volume strategies , 15, 25.α did not differ significantly between young and older women at baseline and did not change consistently in response to any of the low volume HIIT sessions. IGF-1 concentration was reduced in older women; given the specifics of IGF-1-triggered signaling pathways, this may be associated with impaired muscle maintenance and muscle function in the older women. The changes in IGF-1 concentration in response to the HIIT sessions were minor and not significant in any age group, and it seems unlikely that this cytokine is involved in the age related differences (if they exist) in adaptations to low volume HIIT, at least in women.Cytokines are important mediators of various aspects of health and disease, including glucose and lipid metabolism, insulin sensitivity, and skeletal muscle maintenance. We found that the concentration of the inflammatory marker TNF-One limitation of the study is that maximal power was recorded in 5 s intervals, and it is possible that the older women did not achieve maximal power in such a short period. Therefore, the intensity relative to maximal power might have been overestimated in the old women group. In addition, the interventions were not matched by performed work that made it impossible to estimate the effect of exercise intensity per se. One more limitation was the large variability in the blood cytokine responses making it difficult to detect significant changes and conclude how different sprint training protocols affect these markers. Finally, we did not investigate brain mechanisms involved in the present findings while future studies should evaluate brain health markers  response to very low volume HIIT sessions. Furthermore, future research should extend the observations of the current study to male gender and different populations such as athletes, patients, and other groups.Very low volume, high-intensity exercise (6 × 5 s) induces significant physiological stress and seems to be an effective means of training, especially in young women. For older women, more voluminous exercise sessions (3 × 60 s) seem to be more stressful than shorter ones but are still tolerable psychologically. Short maximal intensity exercise bursts can be recommended for both young and older women but practitioners who plan such training programs should consider age effect on the physiological and psychological responses.


The self-assembly of proteins into fibrillar structures called amyloid fibrils underlies the onset and symptoms of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. However, the molecular basis and mechanism of amyloid aggregation are not completely understood. For many amyloidogenic proteins, certain oligomeric intermediates that form in the early aggregation phase appear to be the principal cause of cellular toxicity. Recent computational studies have suggested the importance of nonspecific interactions for the initiation of the oligomerization process prior to the structural conversion steps and template seeding, particularly at low protein concentrations. Here, using advanced single-molecule fluorescence spectroscopy and imaging of a model SH3 domain, we obtained direct evidence that nonspecific aggregates are required in a two-step nucleation mechanism of amyloid aggregation. We identified three different oligomeric types according to their sizes and compactness and performed a full mechanistic study that revealed a mandatory rate-limiting conformational conversion step. We also identified the most cytotoxic species, which may be possible targets for inhibiting and preventing amyloid aggregation. Protein misfolding is the result of an alteration of the normal protein folding process. Incorrectly folded proteins frequently form aggregates with different morphologies, from amorphous structures to highly organized fibrillar aggregates called amyloid plaques. The presence of insoluble amyloid deposits in damaged tissues is a feature of approximately forty neurodegenerative and systemic pathologies of great socioeconomic importance, including Alzheimer’s, Parkinson’s and Huntington’s diseases and diabetes mellitus type 22353supra-physiological concentrations in the μM regime remain too low for some of conventional biophysical techniques. Therefore, studying the processes underlying the initial stages of amyloid aggregation requires techniques with molecular resolution. In particular, innovative computational simulations and single-molecule fluorescence (SMF) are enriching our knowledge of amyloid assembly from a molecular perspective. For example, in silico experiments have suggested the importance of the hydrophobically driven coalescence of monomers prior to conformational conversion into aggregates that are capable of causing amyloid proliferation251517These early stages of aggregation represent a challenge for conventional biophysical techniques because these techniques only provide average information about the ensemble of the population and cannot provide insight into the heterogeneity of the samples. Similarly, conventional biophysical techniques exhibit relatively low sensitivity, setting specific concentration requirements for the measurements. This requirement frequently leads to studies of protein aggregation using high protein concentrations that are usually far from physiologically relevant ranges. For instance, amyloid-β is present at nM levels under physiological conditions8α-spectrin SH3 domain (N47A-SH3), which exhibits enhanced aggregation behaviour compared with the wild type domainOne of the major strategies in protein aggregation research is the use of protein model systems that are not strictly associated with any particular disease but demonstrate aggregation that evolves into amyloid formation, thus representing a valuable tool for drawing generalized conclusions. Among the suitable protein models, SH3 domains have been extensively used in amyloid studies because of their ability to form amyloid aggregates under certain pH, temperature and ionic strength conditionssupra-physiological concentrations.In the present work, we investigated the mechanism of amyloid oligomerization by the N47A-SH3 domain at the molecular level by further evaluating the progress of these aggregates. Our work, which is well supported by other bulk biophysical techniques, provides insights on the intimate mechanism of oligomerization toward the formation of amyloid fibrils. Specifically, we provide experimental evidence for a two-step nucleation mechanism for the formation of aggregates that evolve into mature fibrils at low in silico studies have reported the crucial role of nonspecific contacts as the initial step in aggregationR0, for this pair of fluorophores is 51 ÅE, when the labelled monomers interacted with each otherPrevious studies of the N47A-SH3 aggregate cascade were performed at high (>0.36 mM) protein concentrations to overcome the lag phase, although only a single nucleation step was detected kinetically. However, we sought to investigate the low concentration regime, which emulates the slow aggregation conditions observed in amyloid diseases. Under these conditions, E, with much higher reliability than conventional single-molecule FRET experiments25We studied the early stages of amyloid oligomerization of the N47A-SH3-DA samples in aliquots at different incubation times by analyzing the samples using SMF pulsed interleaved excitation  FRET efficiency, E vs. τA488 correlograms revealed the presence of a population of soluble oligomers at early times as reported previouslyA488. A488 correlograms at different incubation times and different total protein concentrations. These analyses uniquely allowed us to directly identify three types of oligomers and follow their kinetic history upon aggregation. At the very early moments of aggregation, type 1 oligomers exhibited a low FRET efficiency  and small size (4–10 monomer units). In a previous work, we estimated the apparent dissociation equilibrium constant for these type 1 oligomers as 10.1 ± 0.9 nM2) whose size was comparable to the initial population (4–10 monomeric units) but that displayed a high FRET efficiency as evidenced by a reduced τA488 value efficiency , thus supporting a more compact structure of the aggregates. At longer incubation times, a third compact population of large oligomers (from 20 to over 100 monomers) that also exhibits high FRET efficiency clearly arose (type 3). The distinction between types 2 and 3 was not detectable in the E vs. τA488 correlograms because the heterogeneity between populations was only accessible from the different total fluorescence intensities of the events. Importantly, even for fibrillar samples incubated for several days, the supernatant always contained a mixture of oligomers of types 1, 2 and 3. This observation suggests a continuous dynamic equilibrium governing the three states and supports the potential function of mature fibrils as a source of readily formed oligomersThe 1, 2, and 3 and followed their variation with incubation time (2 and 3 was evident even after 1 h of incubation. Samples incubated at lower concentrations (20 and 24 μM) exhibited a slower growth of the compact oligomer forms. With this quantitative information, we performed a thorough kinetic and mechanistic study to understand the roles of these species in amyloid fibril formation.We quantified the relative contribution of each type of oligomer by defining appropriate threshold regions in the correlograms . Using tion time . At the 1) and formation (types 2 and 3) of the oligomers were obtained (0) vs. ln[N47A-SH3], 1 oligomers but (1.8 ± 0.4 SEM) and (3.2 ± 0.7 SEM) for the formation of types 2 and 3, respectively. The limiting step among these processes is the formation of the type 2 oligomers, which exhibited the slowest rates at any concentration tested .We performed a thorough kinetic analysis of the disappearance and formation of the different types of oligomers at the single-molecule level using the method for determining the initial rates at different total protein concentrations see . From thobtained . The appn tested . The app1 and type 2 aggregates. This two-step model for amyloidogenic nucleation implies experimental evidence of the mechanism suggested by Knowles and colleagues for aggregation at low concentrations, which was obtained through computer simulations1 oligomers) for amyloid growth. The necessary conversion step from type 1 to type 2 oligomers that we observed for the N47A-SH3 oligomers and that led to the formation of larger oligomers is also consistent with the kinetic model for α-synuclein aggregation reported by Iljina and colleaguesmaster equation formalisma priori model formalisms were used, the nucleation reaction orders obtained from our experimental data were comparable to the orders observed by Iljina and colleagues. In addition, we obtained evidence showing that the conversion from type 1 oligomers to the more compact type 2 oligomers mainly accounted for a first-order conformational rearrangement. Moreover, Iljina and colleagues suggested first-order kinetics for the final conversion from high FRET-efficiency oligomers to fibrils, whereas our results suggest higher-order kinetics for the conversion from type 2 oligomers to larger type 3 oligomers.Hence, aggregate growth requires two types of nuclei, both type A488). Using this technique, we studied the dynamics of N47A-SH3-DA aggregation at different incubation times and concentrations. The A488 FLIM images clearly showed two important features: the presence of small, high-FRET aggregates and growth into large aggregates. A488, within the detected oligomers at different N47A-SH3-DA concentrations  × 103 min, (1.1 ± 0.3 SEM) × 103 min, and (0.38 ± 0.15 SEM) × 103 min for the incubations at 24, 28 and 32 μM, respectively. At 20 μM, the decrease in τA488 was so slow that it was not possible to fit a single-exponential decay curve.We employed an advanced fluorescence lifetime imaging microscopy with pulsed interleaved excitation scheme  × 10−4 min−1 (A488 in the oligomers obtained by FLIM (9.1 × 10−4 min−1 at 28 μM and 20.0 × 10−4 min−1 at 32 μM). Importantly, this result confirms the correlation between the formation of compact and, hence, high-FRET oligomers and the formation of β-sheet-rich species. Interestingly, the values of the apparent rate constants for the formation of type 2 oligomers, which were obtained from SMF-PIE, ranged from (6.1 × 103) to (11.6 × 103) min−1. These rate constants were slightly higher than the rate constants obtained using the FLIM-PIE and far-UV CD techniques. This result was obtained because we were able to specifically focus on individual type 2 events and discard the contributions from other species using SMF-PIE. This information is inaccessible using ensemble techniques; thus, the rate constant values obtained using FLIM and far-UV CD techniques also contain the contributions of other species . The contributions of these species may cause an apparent slowing of the reaction.The molecular information obtained via SMF-PIE and FLIM imaging is unique compared with that obtained from conventional bulk techniques used in amyloid aggregation studies−4 min−1 . This rah) value was approximately 30 nm, which was significantly larger than the estimated radius of the compact N47A-SH3 domain monomer (1.6 nm)201 aggregates at time 0h and the size distributions  and ATTO 647 N  as described elsewhereSingle-molecule fluorescence fluctuation traces were collected using a time-resolved confocal MicroTime 200 fluorescence microscope system . A detailed description of the instrument can be found in the SI; briefly, it consisted of a SMF confocal microscope equipped with an excitation unit containing two spatially overlapped pulsed lasers  that were alternated on a nanosecond time scale to achieve PIEE), τA488, and the apparent oligomer size were obtained for each oligomer event , the fluorescence intensity of the donor burst, FA488, was corrected by the factor τ0/τA488. This correction takes into account the decrease in the emission intensity of A488 on the oligomers caused by FRET or any other dynamic quenching process. This correction was developed and employed here for the first time. Hence, the apparent oligomer size was estimated as follows:The data were analyzed using custom-coded scripts in SymPhoTime 32 (PicoQuant). The selection and counting of single-molecule oligomers in the coincident and FRET events were considered when a burst was simultaneously detected in the donor and directly excited acceptor traces. The burst-wise FRET efficiency . For the kinetic experiments, changes in the CD signal were monitored at 215 nm in a sample incubated in a 0.1-cm path length cuvette. For the dynamic light scattering (DLS) measurements, we used a DynaPro MS-X  equipped with a Peltier element to control the temperature of the sample. The samples were centrifuged at 14,000 rpm for 30 min to remove preexisting fibrillar forms.3 cells/well. After 48 h of cell culture, 20-μL aliquots of the incubated samples (either unlabelled N47A-SH3 or labelled N47A-SH3-DA) were added directly to the wells. After the cells were incubated with the amyloid oligomers for 24 h, 20% v/v CellTiter-Blue™ reagent was added to the wells and incubated for 2 h at 37 °C. Fluorescence was read in a Glomax®-Multidetection System (Promega). Untreated cell controls and wells with reagents only served as background controls and were analyzed together with the treated cells. At least seven independent repetitions of each data point were performed. Statistical analyses of the cell viability of each population were compared against the 100% value of the untreated control cells using the Wilcoxon signed-rank test  to avoid the requirement for normally distributed populations.The impact of protein aggregation on the viability of human osteosarcoma 143B cells .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Diffusion weighted (DW) cardiovascular magnetic resonance (CMR) has shown great potential to discriminate between healthy and diseased vessel tissue by evaluating the apparent diffusion coefficient (ADC) along the arterial axis. Recently, ex vivo studies on porcine arteries utilizing diffusion tensor imaging (DTI) revealed a circumferential fiber orientation rather than an organization in axial direction, suggesting dominant diffusion perpendicular to the slice direction. In the present study, we propose a method to access tangential and radial diffusion of carotids in vivo by utilizing a pulse sequence that enables high resolution DW imaging in combination with a two-dimensional (2D) diffusion gradient direction sampling scheme perpendicular to the longitudinal axis of the artery.High resolution DTI of 12 healthy male volunteers (age: 25–60 years) was performed on one selected axial slice using a read-out segmented EPI (rs-EPI) sequence on a 3T MR scanner.P < 0.01) linear relationship with an adjusted R2 of 0.52. In addition, a linear trend (P < 0.1) could be observed between radial diffusivity (RD) and age.It was found consistently for all 12 volunteers, that the tangential component as the principle direction of diffusion. Mean vessel wall fractional anisotropy (FA) values ranged from 0.7 for the youngest to 0.56 for the oldest participant. Linear regression analysis between the FA values and volunteers age revealed a highly significant (These results point to FA being a sensitive parameter able to capture changes in the vascular architecture with age. In detail, the data demonstrate a decrease in FA with advancing age indicating possible alterations of tissue microstructural integrity. Moreover, analyzing 2D diffusion tensor directions is sufficient and applicable in a clinical setup concerning the overall scan time. Cardiovascular diseases (CVDs) remain the global killer number one. Estimations from the world health organization (WHO) revealed, that about one third of global deaths were attributable to this life threatening diseases in 2012 [Imaging modalities, such as ultrasound, computed tomography (CT), cardiovascular magnetic resonance (CMR) can assist in the detection and classification of morphological pathologies. However, these imaging techniques are applied mainly to determine the degree of luminal narrowing, which is an indispensable information, but a limited biomarker to determine the true impact on future cardiovascular events . A more Recently, diffusion-weighted (DW) CMR has shown great potential to discriminate between normal and pathological vessel tissue by evaluating the apparent diffusion coefficient (ADC) –8 for a Two major constraints prevent the application of DTI to visualize diffusion in the carotid artery wall in vivo. Firstly, the low spatial resolution when using a conventional single-shot EPI (ss-EPI) and a significant loss of SNR at higher spatial resolution as a result of the prolonged echo time. Secondly, the scan time that dramatically increases with the number of diffusion sensitizing gradient directions.In this work we propose a method to overcome these limitations in order to measure tangential and radial diffusion, evaluated from a two-dimensional diffusion tensor, in the carotid artery wall in vivo. High spatial resolution was achieved by utilizing a read-out segmented EPI (rs-EPI) pulse sequence . Due to In this proof of concept we demonstrate the feasibility of measuring diffusion in the vessel wall of carotid arteries in vivo in a non-destructive and non-invasive fashion and provide initial results from 12 healthy male volunteers.The basic idea to reduce the amount of diffusion sensitizing gradients is the reduction of a three-dimensional diffusion measurement to a two-dimensional diffusion measurement perpendicular to the artery’s axis Fig. . This stDWI of a slaughterhouse harvested porcine aorta was performed on one selected axial slice using rs-EPI and both a three-dimensional gradient direction schema with 30 directions  and a two-dimensional schema with 18 directions (acquisition time = ~30 min). Sequence parameters were as follows: FOV = 111 × 111 mm2, matrix = 308 × 308, slice thickness = 10 mm, TR = 2500 ms, TE = 81 ms, GRAPPA = 2, number of readout segments = 31, b-values = 0 and 1000 s/mm2. To extract angle distributions of the principle 3D-DTI tangential vector with respect to the longitudinal axis of the artery Eq. (1) was used.Ex vivo To optimize imaging conditions, ex vivo specimens were immersed in a fluid  having no MR signal and to minimize magnetic susceptibility effects at air-tissue boundaries , 13.The 2D diffusion acquisition scheme was used for in vivo measurements requiring that the diffusion sensitizing directions, lying within a plane in space, are oriented perpendicular to the artery’s axis. This was achieved by defining the diffusion directions in the  coordinate system that is linked to the orientation of the field of view.High resolution diffusion weighted imaging (DW) of 12 healthy male volunteers (25–60 years) was performed on one selected axial slice using rs-EPI on a 3T whole body MR scanner  with a 2 × 4 channel multifunctional coil  and FOV = 189 × 189 mm2, matrix = 346 × 346, slice thickness = 10 mm, TR = 2RR intervals, trigger delay = 50 ms to acquire images in the diastolic phase, TE = 93 ms, GRAPPA = 2, number of readout segments = 9, b-values = 0, 200, 400, 600 s/mm2, acquisition time = ~12 min. Eighteen gradient directions were defined on a hemicycle within a plane oriented perpendicular to the longitudinal axis of the carotid artery. To minimize artifacts, introduced by pulsatile vessel motion, peripheral pulse-triggering was performed using the systems’ internal physiological monitoring unit. To avoid chemical shift artifacts caused by peri-adventitial fat, a spectral fat saturation pulse was applied. A series of two-dimensional (2D) angiographic axial vessel scout images  preceded the main task to locate the carotids and orient the slice perpendicular to the long axis of the artery. Measured DWI data with an in-plane resolution of 0.55 × 0.55 mm2 were interpolated to 0.2 × 0.2 mm2 using zero-filling.Registration of the individual DW images was achieved by applying publicly available elastix software [software , 15. In software , 17.The study was approved by the institutional review board (IRB) as well as followed Declaration of Helsinki recommendations and informed consent was signed by each participant prior to the MR measurements.To test the robustness of repeated FA measurements using the above introduced in vivo high resolution DW sequence, the carotid artery of four male volunteers was repeatedly  measured and the coefficient of variation (CV), defined as the ratio of the standard deviation (σ) to the mean (x̅), was calculated.2 (in steps of 200 s/mm2) for a certain diffusion gradient direction perpendicular to the slice direction.The afore mentioned in vivo high resolution diffusion weighted imaging (DWI) parameter setup was used to investigate optimal b-values on one selected axial carotid slice of a male volunteer. With all other sequence parameters held constant, the TE was set to 97 ms in order to allow for measurements with increasing b-values from 0 to 1000 s/mm1) data with volunteer’s age a linear regression analysis was used to estimate parameters (coefficient of determination (R2), slope and intercept) and assess linearity. In addition, 95 % confidence and prediction intervals for the linear regression were calculated. An F-test for linear models was employed to compare variances and a probability value of P < 0.05 was considered as significant. Normal distribution was tested based on Shapiro-Wilk Normality Test. All analyses were performed using freely available R-project software [To investigate the relationship between fractional anisotropy (FA), radial diffusivity (RD) and primary eigenvalue (λsoftware .2) sensitizing it to water diffusion. S0 represents the reference signal without diffusion weighting (no diffusion gradient).The 3D diffusion tensor described by a symmetric 3 × 3 matrix and its derived quantities can easily be reduced to the 2D case where the tensor is defined by a 2 × 2 matrix. Mathematical aspects are discussed in the following and are detailed described by Kingsley et al. –21. A Led by solving Eq. (3):Y represents the calculated apparent diffusion coefficients (18 element row vector), H is derived from the normalized gradient components Eq. (4) and d is the tensor given as a three-element column vector Eq. (5).The 2D gradient direction sampling scheme enables the evaluation of the diffusion tensor d corresponding eigenvalues (λ) and eigenvectors (ε) can be calculated on a pixel by pixel basis using eigenvalue decomposition. To study tangential and radial diffusion of the vessel wall 2D FA values were obtained using Eq. (6).From tensor 1), RD  and the mean diffusivity (MD) which is the average of the eigenvalues.FA values close to zero mean no directed diffusion whereas anisotropy is given if one of the eigenvalues will be higher than the other. Calculation included the primary eigenvalue . It can be observed that the signal is suppressed in the direction of the diffusion sensitizing gradient, marked by the white arrowhead. Calculating the 2D diffusion tensor revealed consistently for all 12 volunteers that the largest eigenvalue is oriented tangential to the vessel wall while the second component therefore shows a radial orientation. This fact provides clear evidence for the tangential direction as the principle contribution for diffusion within the vessel wall as shown in Fig. Table Table 1 and λ2. For the 3D case the tangential component corresponds to the largest eigenvalue (λ1). The radial and longitudinal components can either be λ2 or λ3 because both eigenvalues are in a comparable range of magnitude  for a certain diffusion direction perpendicular to the slice direction . In addition, outlined in Fig. 2) and the maximum difference of both populations found at a b-value of about 605 s/mm2. In addition to the diffusion decay factor e(−bD), signal loss is given by the vessel’s tissue T2 relaxation time and predefined echo time as expressed by the decay constant e(−TE/T2). All these effects contribute to a signal drop and justify a distribution of selected b-values between zero and the found maximum.To estimate optimal b-values and study the tissues’ anisotropic properties, an in vivo vessel wall ADC map was generated from multiple measurements with increasing b-values  data for 12 male volunteers ranging from 0.7 for the youngest to 0.56 for the oldest are plotted as a function of age. The linear regression for the FA and age relationship was highly significant P < 0.01 with an adjusted R2 of 0.52 and the linear equation y = −0.0049x + 0.79. For the relationship between mean RD and age a linear trend P < 0.1 with an adjusted R2 of 0.18 was observed. No linear relationship P > 0.6 between the mean primary eigenvalues (λ1) and age was found. This analysis indicates that FA, measured within the vessel wall, is a sensitive parameter able to capture changes in the vascular architecture due to aging.In Fig. In this work, we demonstrated the feasibility of measuring diffusion anisotropy in human carotids in vivo in a non-destructive and non-invasive fashion. In contrast to previous works that aim for quantitative diffusion imaging in vivo  along the slice direction (z-direction) our results suggests that tangential and radial diffusion is accessible by using a pulse sequence that enables high resolution DW imaging measurements in combination with a 2D gradient direction sampling-scheme orientated perpendicular to the vessel’s longitudinal axis. Furthermore, our in vivo and ex vivo results corroborate the fact that the principle diffusion direction is aligned perpendicular to the long axis of the artery as demonstrated by ex vivo studies on porcine aortas , 10. Mor1 indicate a rather constant behavior with advancing age. These indicators taken together may provide complex information about ongoing processes on the vascular architecture. However, the reported aging effect is notable but lifestyle may also influence vascular wall structure and was not covered by this proof of concept. To investigate this process more closely, further experiments are needed including more subjects. Another interesting application of the proposed method might be to study the influence of plaque components on the diffusion process. Future work on vessel wall diffusion imaging may help to understand the formation of such a complex disease.Recent investigations of ADC values along the z-direction (slice direction) were done on healthy volunteers and patients but the relevance of aging was not considered –8. In coTo achieve accurate estimates of the self-diffusion tensor the sensitivity to motion-induced phase errors has to be reduced to a minimum. Motion is a severe factor in diffusion imaging in general and specifically when trying to access diffusion in the carotid wall , 8. SeveConventional single-shot EPI’s are fast sequences that enable the acquisition of a slice within a single repetition time but suffer from susceptibility artifacts and T2* blurring due to the prolonged time required to fill the k-space, low resolution and significant loss of SNR at higher resolution. Diffusion tensor imaging is inherently a technique suffering from low SNR. High spatial resolution cannot be achieved using such conventional ss-EPI due to the signal loss as a result of the prolonged echo time. Hence the use of rs-EPI is a prerequisite for high spatial resolution. It can be seen from the representative diffusion weighted images of Fig. In conclusion, we present a novel method to access tangential and radial diffusion of carotids in vivo by utilizing a pulse sequence that enables high resolution DW imaging in combination with a 2D diffusion gradient direction sampling scheme perpendicular to the longitudinal axis of the artery. In contrast to standard DW methods evaluating the ADC for a certain vascular segment along the artery’s axis our method allows to image the water diffusion tensor within the vessel wall with a high spatial resolution. Ex vivo studies on porcine arteries suggested a dominant circumferential fiber orientation rather than an organization in axial direction supporting our 2D approach. Furthermore, our results demonstrate a decrease in fractional anisotropy (FA) with advancing age indicating FA as a sensitive parameter able to capture changes in the vascular architecture with age. Moreover, analyzing 2D diffusion tensor directions is sufficient and applicable in a clinical setup concerning the overall scan time. Our data demonstrate for the first time the feasibility of measuring diffusion anisotropy in human carotids in vivo in a non-destructive and non-invasive fashion. Future work will investigate the influence of plaque components on the diffusion process, which may help to understand the formation of a complex disease.


Chemical vapor deposition grown graphene was transferred to BN/sapphire substrates for evaluation of more than 30 samples using Raman and Hall effects measurements. A more than 2x increase in Hall mobility and 10x reduction in sheet carrier density was measured for graphene on BN/sapphire compared to sapphire substrates. Through control of the MOCVD process, BN films with roughness ranging from <0.1 nm to >1 nm were grown and used to study the effects of substrate roughness on graphene transport. Arrays of graphene field effect transistors were fabricated on 2” BN/sapphire substrates demonstrating scalability and device performance enhancement.Wafer scale (2”) BN grown by metal organic chemical vapor deposition (MOCVD) on sapphire was examined as a weakly interacting dielectric substrate for graphene, demonstrating improved transport properties over conventional sapphire and SiO Unfortunately, interactions between graphene and its environment, including the substrate, greatly degrade its transport properties. Substrate induced scattering from charged impurities, roughness, and phonons will reduce mobility3. The most successful approach to mitigate these effects has been the use of a thin van der Walls (vdW) buffer layer, like hBN5 or MoS26, preserving the mobility of the graphene to values near those of suspended material. Of the available vdW materials, hBN is one of the only insulators and due to is dielectric properties (comparable to SiO27), high surface optical phonon energy (169 eV)8 and atomically smooth surface; it has been the most successful and widely used material for preserving and protecting the intrinsic properties of 2D materials 14. The overwhelming majority of this work has used hBN flakes exfoliated from high quality bulk materials. The high purity and crystal quality of this exfoliated material has given rise to amazing properties; however, due to the small size, low yield and poor reproducibility of these exfoliated flakes this process is not scalable or suitable for device fabrication beyond the laboratory.Due to its amazing properties, graphene continues to be of great interest more than a decade after its experimental discovery18 and insulating substrates22. Taking advantage of the catalytic nature of metal substrates, like Cu and Ni, high quality material can be grown at quite low temperatures (<1000 °C). Since this process it typically surface catalyzed the controlled growth of mono-to few-layer hBN is possible23. The hBN grown on metal films can then be transferred to insulating substrates for further processing and characterization. Improved transport in graphene has been demonstrated using such transferred hBN films25 as compared to conventional substrates. Lee et al.24 demonstrated a 3x increase in graphene mobility when a few layer hBN buffer was inserted on SiO2/Si. Back gated graphene field effect transistors (FET) on hBN, grown on Fe substrates showed improved mobility and reduced intrinsic doping as compared to similar devices directly on SiO2/Si25. For many of the applications described above hBN grown on a metal will inevitably require transfer to insulating substrates adding complexity, sources of contamination, and damage from the fabrication process.The need for large area high quality hBN thin films for 2D substrates, dielectrics, and tunneling barriers has generated considerable upswing in hBN growth. High quality hBN thin films from mono to many layers thick over large areas have been produced using chemical vapor deposition (CVD) using both metallic27. The formation of large hexagonal wrinkles has also been observed in BN grown on sapphire due to the thermal expansion mismatch between BN and the substrate28. These features make BN films grown on sapphire distinct from those on metals. Jang et al. reported growth of smooth few-layer hBN on sapphire and demonstrated a 2x increase in graphene field effect mobility as compared to graphene on SiO2/Si29. Now with the availability of high quality large area CVD grown BN films on sapphire, more work is needed to explore their applications as substrates and dielectrics for 2D electronics.Alternatively, hBN films grown directly on insulating substrates do not require transfer prior to device processing. Due to the challenges of growing hBN directly on an insulating substrate, such as sapphire, it has been much less explored than its counterpart grown on metals. Growing on sapphire it is much more challenging to produce continuous ultra-thin films of mono– or bi-layer thickness, and as such, most reports demonstrate films from few to tens of nanometers thick2-BN on sapphire as a weakly interacting substrate for graphene. The basal plane of sp2 bonded BN forms the same honeycomb lattice as graphene and can form atomically thin and smooth layers. In multilayer BN, layers are known to stack hexagonal (AB stacking), rhombohedral (ABC stacking), or turbostratic (random stacking) with very small differences in inter planar spacing. For this work, CVD grown graphene was transferred to wafer scale sp2 BN/sapphire (now referred to as BN) grown by metal organic chemical vapor deposition (MOCVD) to study the effects on transport properties by Hall and FET device measurements. The effect of BN roughness was also investigated using BN films with roughness from <0.1 nm to >1 nm. Results show a more than 2x increase in graphene mobility when transferred to BN on sapphire substrate with a strong dependence on substrate induced doping related to BN roughness.In this paper, we investigate the use of few-layer sp26. Reducing the V/III ratio or increasing the pressure disrupts these self-terminating conditions resulting in thicker, rougher (>1 nm) films with a random orientation . From XPS analysis, the B/N was measured to be 1/1 to 1.05 across a range of samples. To demonstrate uniformity across each wafer Fig. 2-BN E2g mode at 5 points across 2” BN/sapphires wafers. The atomically smooth BN/sapphire is well suited for application as a substrate for 2D materials, while rougher films are useful to study the effects of substrate roughness on graphene transport.For application as a graphene substrate, BN films need to be atomically smooth, atomically flat, insulating and uniform over large areas. Growth of BN on sapphire by MOCVD at low pressure (20 Torr) and high V/III ratios (>2250) has been shown to produce films fitting these parameters with excellent thickness uniformity (1.6 + /−0.1 nm) and low roughness (<0.1 nm) over large areas Fig. . Under tion Fig. 30. Varyi2/Si as reference substrates. Raman spectroscopy of graphene films was used to compare the different dielectric substrates. Figure D/IG) taken from multiple spots on each sample was used for comparison, Fig. D/IG is 0.09 with a standard deviation (STD) of 0.05 across 6 samples. Comparable values of ID/IG of 0.1 with a STD of 0.05 were found for the samples transferred onto both the smooth and rough BN/Sapphire substrates indicating that the graphene is both uniform and undamaged during the transfer process regardless of the substrate.Monolayer graphene films grown by CVD on Cu were transferred to 20 different BN on sapphire substrates with BN roughness ranging from >1 nm to <0.1 nm to study the effects of BN roughness and morphology on graphene. Half of the BN/sapphire substrates were grown under self-terminating conditions and have measured roughness <0.1 nm. Graphene was also transferred to sapphire and SiOH) as a function of sheet carrier density (ns) for each sample. As a comparison, graphene transferred to c-plane sapphire and SiO2/Si substrates was also investigated. From this data a clear separation into groups based on the substrate can be observed, illustrating the impact the underlying substrate has on transport. Graphene films on sapphire and SiO2/Si have the lowest µH and highest ns. By switching to even a very rough (>1 nm) BN/sapphire substrate a noticeable reduction in ns and increase in µH is achieved. This trend of improving graphene transport continues as the BN roughness is reduced. A 2x increase in µH and one order of magnitude decrease in ns was achieved on the smoothest BN/sapphire substrates. The substrate is known to effect ns and µH in a number of ways causing doping, phonon scattering and/or corrugation of the graphene layer. At the surface of SiO2 and sapphire hydroxyl groups can form32, which will adsorb water and p-type dope the graphene34. Switching to the inert vdW surface of BN will eliminate this substrate doping effect assuming a near perfect surface and interface. For graphene supported by MOCVD BN/sapphire we expect a contribution to ns from surface impurities and defects, which may form hydroxyl groups similar to sapphire and SiO2. As such, it is not surprising to see a higher ns in graphene supported by rough BN. If we compare the morphologies and layer orientation  phonons3 from the substrate as well as graphene longitudinal acoustic (LA) phonon2 and short range scattering39 will all effect mobility. The mobility limited by Coulomb scattering from impurities, short range scattering from defects and roughness are all temperature independent mechanisms that will be defined here as µ0. The temperature dependent mechanisms for the graphene LA phonon (µAL) and substrate SO phonon (µSO) can be expressed as:ρsis the graphene 2D mass density, vf is the fermi velocity, vs is velocity of sound, DA is the acoustic deformation potential, E0 is the substrate phonon energy, and C is the coupling strength. Using Matthiessen’s rule, the total limited mobility is expressed as 1/µtot = 1/µ0 + 1/µLA + 1/µSO. Accordingly, we observe an inverse mobility dependence on ns 8 and sapphire (55 meV)40 shows that switching to a BN/sapphire substrate can positively impact µH at room temperature.For substrate supported graphene, scattering due to the surface impuritiess to be independent of temperature over the range of 10 to 320 K −1 shows good agreement with graphene on both smooth BN and sapphire. Only µ0, DA, C, and E0 were obtained from fitting given in Fig. A were determined to be 17.7 and 16 eV for sapphire and smooth BN substrates in good agreement with theoretical and experimental values2. The experimentally determined values for E0 are 135 meV for the smooth BN/sapphire and 60 meV sapphire. On a rough BN/sapphire substrate, graphene mobility is not well described by Eqns. . Since at higher temperatures mobility is limited by substrate induced SO phonon scattering, we fit the graphene on rough BN mobility to µtot = (1/µ0 + 1/µSO)−1 for temperatures greater than 150 K. This fit gives us an estimate of E0 (140 meV) for these BN/sapphire substrates, which is in good agreement with the smooth BN/sapphire. Using this modified version of µtot we also obtain a value of 1851 cm2/Vs for µ0, which is between those for the smooth BN and sapphire. Although, this term contains the contribution of roughness to µtot we cannot explicitly determine its contribution considering Coulomb and short range scattering are also temperature independent. If we qualitatively compare µ0 from the graphene on smooth BN and sapphire samples, which have similar roughness (<0.1 nm), we find the effects of impurity and carrier density (Coulomb and short range) to be significant suggesting a stronger contribution to µtot than roughness.Temperature dependent mobility for graphene on sapphire, rough BN, and smooth BN are shown in Fig. 2/Si FETs were also fabricated and tested. Typical Id-Vd measurements showing gate modulation in devices on smooth and rough BN/sapphire are shown in Fig. 2O3 has been shown to dope graphene and can significantly increase electron concentration, it is expected that the deposition of the Al2O3 top gate is related to this difference in majority carrier type44. The observed switch (from p to n) in majority carrier type in devices on smooth BN/graphene, but not rough BN/sapphire, is due to the lower background carrier density.Further quantifying MOCVD grown BN as a substrate for graphene devices, top-gated FETs were fabricated on a 1” graphene film transferred to a 2” BN/sapphire substrate Fig. . The mas2/Si were also studied. The Dirac point voltage is clearly different for these three dielectric substrates, Fig. 45. In our case, the bottom BN layer in the devices presented here also changes the Dirac point. Figure 46.The transfer characteristics of gFET devices on rough BN/sapphire, smooth BN/sapphire and SiOm is transconductance, defined as gm = dId/dVg, Cox is the gate oxide capacitance (Cox = κε0/tox), W is channel width, and L channel length. Using this method, we found mobility depended strongly on the channel length  graphene FET arrays were fabricated on 2” BN/sapphire and investigated showing good uniformity across the array. This collective work demonstrates significant performance improvements in graphene using scalable methods for producing BN/sapphire substrates.We have presented results from a detailed study on MOCVD BN on sapphire as a weakly interacting dielectric substrate for graphene. Wafer scale BN can be grown on sapphire with low roughness and excellent uniformity. Using Raman, Hall effect, and FET device characterization we studied the impact the dielectric substrate has on CVD transferred graphene. A greater than 10x decrease in graphene carrier density and 2x increase in mobility is achieved by using a BN/sapphire substrate as compared to SiO3 and TEB precursor gases used for N and B, respectively, are injected such that they are not heated nor do they interact with one another until they reach the surface of the substrate. The reactor was kept at a pressure of 20 Torr and the substrate was heated to 1000 °C during the growth. The ratio of NH3 to TEB was set between 2250 to 950 to achieve the desired morphology and quality of BN films.Boron Nitride thin films used in this study were grown via a metalorganic chemical vapor deposition (MOCVD) technique. The substrates used for BN growth were c-plane sapphire which allows for uniform, few layer BN to be grown. In a cold-walled vertical MOCVD reactor, the NH50. After transfer, graphene films were annealed in forming gas (5% H2: 95% Ar) for 1 hr at 300 °C to remove any additional residue from the transfer process.Graphene was grown on a copper substrate by CVD using methane as the carbon source. Films were subsequently transferred to dielectric substrates using a typical wet transfer with PMMA, similar to the process described in ref.in-situ FIB lift out technique on an FEI Dual Beam FIB/SEM and imaged with a FEI Tecnai TF-20 FEG/TEM operated at 200 kV in high-resolution (HR) TEM mode. The surface morphologies of the graphene and BN films were analyzed by AFM using a Bruker Dimension Icon in tapping mode. Raman measurements were performed using a Renishaw inVia system under a backscattering geometry with scattered radiation collected along the sapphire [0001] direction. A 4 mW 488 nm excitation source, 20 µm slits, and a 3000 l/mm grating was used for each measurement. The transport properties were characterized by Hall-effect measurements. For room temperature measurements, an Accent 5500 system was used under ambient conditions. For temperature dependent measurements from 10 to 320 K, a Lakeshore 7507 system was used. Prior to measurement the sample was loaded into the cryostat, heated to 350 K under a He atmosphere and held overnight. Measurement sweeps were taken during cooling to 10 K and heating to insure a consistent measurement. If samples were not heated to 350 K, the measurements were found to be quite different on heating and cooling.Transferred graphene and BN MOCVD layers were characterized using a suite of techniques. The BN thickness was measured by X-ray reflectance (XRR) using a PANalytical Empyrean X-ray diffractometer at a grazing incidence angle. Cross section TEM samples were prepared using the 2/Si substrates. Graphene mesas were defined using O2-plasma etch followed by source/drain metal contacts (Ti/Pt/Au) using a first lift-off process. A 20-nm thick Al2O3 was used as gate dielectric. Finally, 100-nm thick gate metal (Ti/Au) contacts were defined using a second lift-off process. Gate dimensions were chosen such that electron beam lithography process is not required for the FET fabrication process. An Agilent 4156 C precision semiconductor parametric analyzer was used for all the electrical characterization of the FETs.Top-gated graphene FETs were fabricated on transferred graphene on BN/sapphire and SiOSupplementary information


High-carotenoid corn (Carolight®) has been developed as a vehicle to deliver pro-vitamin A in the diet and thus address vitamin A deficiency in at-risk populations in developing countries. Like any other novel crop, the performance of Carolight® must be tested in different environments to ensure that optimal yields and productivity are maintained, particularly in this case to ensure that the engineered metabolic pathway does not attract a yield penalty. Here we compared the performance of Carolight® with its near isogenic white corn inbred parental line under greenhouse and field conditions, and monitored the stability of the introduced trait. We found that Carolight® was indistinguishable from its near isogenic line in terms of agronomic performance, particularly grain yield and its main components. We also established experimentally that the functionality of the introduced trait was indistinguishable when plants were grown in a controlled environment or in the field. Such thorough characterization under different agronomic conditions is rarely performed even for first-generation traits such as herbicide tolerance and pest resistance, and certainly not for complex second-generation traits such as the metabolic remodeling in the Carolight® variety. Our results therefore indicate that Carolight® can now be incorporated into breeding lines to generate hybrids with locally adapted varieties for further product development and assessment. Plant carotenoids accumulate in plastids and play fundamental roles in photosynthesis as accessory pigments and in the xanthophyll cycle by preventing photo-oxidative damage3de novo. Furthermore, carotenoids such as lycopene provide general health-promoting antioxidant activity whereas lutein and zeaxanthin specifically help to protect the retinal cells of the macula against phototoxic damage, thus preventing age-related macular degeneration (ARMD)Carotenoids are essential pigments found mainly in plants and microbespsy1 and Pantoea ananatis crtI. Golden Rice II produces 37 μg g−1 dry weight (DW) of total carotenoids (26-fold more than the near isogenic wild-type variety) and Carolight® corn produces 84 μg g−1 DW of total carotenoids (156-fold more than the near isogenic wild-type variety)68912Several transgenic crops with high carotenoid levels have been generated by overexpressing carotenogenic genes, including Golden Rice IIWe therefore tested the hypothesis that Carolight® is similar in terms of agronomic performance to its non-transgenic near isogenic counterpart when compared in a controlled environment and under field conditions. The key objectives of our studies were (i) to determine whether the accumulation of carotenoids in Carolight® affects the rates of photosynthesis, biomass accumulation and partitioning into different organs in experimental field trials (study 1); and (ii) to compare the effectiveness of the introduced trait in a controlled greenhouse environment and the field (study 2). In order to broaden the range of environments and to test the consistency of adaptation and performance between Carolight® and its near isogenic counterpart, we compared both genotypes in field plots under different soil nitrogen (N) availability regimes and contrasting source-sink relationships during grain filing. N availability is known to influence photosynthesis and biomass accumulation17Zea mays) near-isogenic genotypes: the wild-type white endosperm variety M37W and the high-carotenoid transgenic line Carolight®, expressing corn psy1 and Pantoea annatis crtI in a M37W genetic background. M37W seeds were obtained from CSIR . Seeds were planted in the experimental fields of the School of Agronomy, University of Lleida, Spain  (study 1) or in pots maintained in the greenhouse (study 2).We used two corn (0 = 0 kg ha−1 and N200 = 200 kg ha−1. The N was applied as urea at the V6 stage (six fully-expanded leaves). Source–sink relationships were manipulated at silking (the R1 stage) by removing most of the leaves, and tests were carried out using plants that were distributed uniformly at the same density and visually identical in terms of size, leaf number and developmental stage. Most of the leaf laminae were removed by cutting the leaves on the collar, between the lamina and sheath. Only the two leaves adjacent to the ear were left on the plant (those immediately below and above). The effects of all treatments and their interactions were tested for statistical significance by the analysis of variance (ANOVA).Two different fertilization regimes were applied: N0 and N200) with subplots consisting of source–sink treatments. Plots were randomized with four replicates, each consisting of six rows, 70 cm apart and 6.47 m in length. Each plot was fully irrigated. Pests, diseases and weeds were controlled or avoided by spraying recommended insecticides, fungicides and herbicides at the doses suggested by their manufacturers whenever necessary.In study 1, seeds were sown in the field on 5 May 2013 and treatments consisted of a factorial combination of the two maize genotypes and two N treatments , and the dissected endosperm was frozen in liquid nitrogen and stored at −80 °C.8 (eight fully-expanded leaves), R1 (silking) and R6 (maturity)8 and R1 using a Li-3100C area meter . All plant materials were oven dried for 72 h at 65 °C after processing and weighed to determine the dry biomass of each fraction.Three developmental stages were chosen for biomass sampling: VAfter weighing, all plant materials were ground in analytical mills, and the N concentration was determined using the Kjeldahl method. The specific leaf area (SLA) was calculated by dividing the leaf area per plant by leaf mass, and the specific leaf nitrogen (SLN) content was calculated as the ratio of leaf nitrogen content to leaf area. The leaf area index (LAI) was calculated as the total green area of leaf tissue per unit ground area. N utilization efficiency (NUtE) was calculated as the ratio between yield and N uptake at physiological maturity. The N harvest index (NHI) was calculated as the ratio between N content in the grains and aboveground biomass at maturity.8 until grain filling was complete, using a LCi portable photosynthesis system  which measures net CO2 assimilation (net photosynthetic rate). Measurements were taken using the last fully-expanded leaf between mid-morning and noon on cloudless days by holding the photosynthetic chamber perpendicular to the direction of incident solar radiation. The leaf chlorophyll concentration from V8 to R6 was estimated in situ using a SPAD-520 portable chlorophyll meter  on the same leaves used to measure gas exchange. The duration of photosynthesis was calculated by integrating values across the growth cycle and measuring the area underneath the curvePhotosynthetic rates and related traits were determined for individual leaves every 2 weeks from VTotal RNA was isolated using the RNeasy Plant Mini Kit  and DNA was removed with DNase I . Total RNA was quantified using a Nanodrop 1000 spectrophotometer , and 2 μg total RNA was used as the template for first strand cDNA synthesis with Ominiscript reverse transcriptase (Qiagen) in a 20-μl total reaction volume, following the manufacturer’s recommendations.Zmbch1, Zmbch2, Zmcrtiso, Zmcyp97a, Zmcyp97b, Zmcyp97c, Zmlycb, Zmlyce, Zmpds, Zmzds, PacrtI and Zmpsy1 gene expression in a BioRad CFX96 system with 25-μl reaction mixtures containing 10 ng cDNA, 1x iQ SYBR Green Supermix  and 0.2 μM forward and reverse primers21Quantitative real-time RT-PCR was used to analyze 2 at 37 °C, flushed with argon and stored at −80 °C.Maize endosperm was excised by removing the seed coat and embryo. Samples were freeze-dried before extraction and were ground to a fine powder. Carotenoids in 50–100 mg samples were extracted in 15 ml methanol:ethyl acetate (6:4 v/v) at 58 °C for 20 min. The mixture was filtered and transferred to a separation funnel before we added 15 ml hexane:diethyl ether (9:1 v/v) and agitated gently for 1 min. We then added 15 ml saturated NaCl, the aqueous phase was removed, and the organic phase was washed twice with water. The samples were dried under NThe extracts were dissolved in 210–600 μl injection solvent, which was three parts acetonitrile/methanol (7:3 v/v) to two parts acetone. Ultra-high-performance liquid chromatography (UHPLC) analysis was carried out at SCT-DATCEM, University of Lleida, using an Acquity Ultra Performance LC system linked to a photodiode array (PDA) 2996 detector . Mass detection was achieved using an Acquity TQD tandem quadrupole mass spectrometer equipped with a Z-spray electrospray interface (Waters). MassLynx software v4.1 (Waters) was used to control the instruments and also for data acquisition and processing. UHPLC separations were carried out on an Acquity UPLC C18 BEH 130 Å, 1.7 μm, 2.1 × 150 mm reversed-phase column (Waters). The mobile phase consisted of solvent A = acetonitrile/methanol (7:3 v/v) and solvent B = 100% water. Carotenoids were quantified using a PDA detector and identified as previously described200 treatment than the N0 treatment (The chlorophyll content of leaves (i) declined during plant growth from 43.8 ± 2.7 SPAD units at 70 DAS to 26.1 ± 2.0 SPAD units at 141 DAS and (ii) was slightly higher under the Nreatment . No consreatment .2 m−2 s−1 at V8 and slowly declining to 13.7 ± 0.4 mmol CO2 m−2 s−1 at maturity, but soil N availability did not affect the leaf photosynthetic rate. Both genotypes exhibited similar photosynthetic rates across treatments and throughout development. Consequently, the photosynthetic rate averaged across time and N levels was almost identical for both genotypes  but there were no genotype-specific differences at either developmental stage under the N200 treatment  than to average grain weight . There was no significant difference in grain number between genotypes, but there was a tendency towards a higher grain number in Carolight® than wild-type corn: 1895 ± 247 vs 1656 ± 126 grains m−2 under the N0 treatment and 1807 ± 378 vs 1554 ± 72 grains m−2 under the N200 treatment  than in the defoliated plants (2.05 ± 0.01%). This may have occurred because defoliation also reduced the grain number by ~25% in both genotypes.Zmpsy1 and PacrtI expression levels in the endosperm of plants grown in the field or in the greenhouse were measured by quantitative real-time RT-PCR at four time points, corresponding to four different endosperm developmental stages during grain filling: 15, 20, 25 and 30 days after pollination (DAP). Both transgenes were expressed in both environments, and in both cases PacrtI mRNA accumulated at three-fold lower levels than Zmpsy1 mRNA at all time points. The expression profile of PacrtI was similar under field and greenhouse conditions (Zmpsy1 mRNA levels in young endosperm (15 DAP) for plants growing in the greenhouse compared to plants in the field, but the expression levels in the two environments converged during seed development (nditions . There welopment .Zmbch1), β-carotene hydroxylase 2 (Zmbch2), carotene isomerase (Zmcrtiso), carotene ε-hydroxylase , lycopene β-cyclase (Zmlycb), lycopene ε-cyclase (Zmlyce), phytoene desaturase (Zmpds) and ζ-carotene desaturase (Zmzds). No significant differences in the expression levels of these endogenous carotenogenic genes were observed between the two environments, and the abundance of each transcript did not change substantially over time . The prevalent carotenoids were phytoene, zeaxanthin, lutein, β-carotene, α-cryptoxanthin, β-cryptoxanthin and antheraxanthin. The maximum total carotenoid accumulation occurred at ~40 DAP in both environments, and was 113 ± 7.5 μg/g DW for plants in the greenhouse compared to 99 ± 2.7 μg/g DW for plants in the field. However, no significant differences in the total carotenoid content were observed in the mature kernels at 60 DAP. The total carotenoid content was 96.8 ± 3.7 μg ghe field . M37W plvs 25% for greenhouse plants), antheraxanthin (11% vs 5%) and β-cryptoxanthin (8% vs 5%). Indeed, higher proportions of these carotenoids were detected in field-grown plants compared to their counterparts in the greenhouse, with corresponding lower amounts of β-carotene  than field-grown plants    and 6b.Novel crops with enhanced traits should be tested against their near-isogenic wild-type counterparts under a variety of environmental conditions to ensure that performance is maintained in the environments that are likely to be most relevant for commercial deployment. However, it is rare for such comprehensive analysis to be performed even for the simplest first-generation input traits such as herbicide tolerance and pest resistance, and unprecedented for second-generation output traits such as enhanced metabolic pathways. This is particularly the case for products with humanitarian applications developed outside the private sector. We therefore addressed the hypothesis that a nutritionally enhanced crop such as Carolight® should be similar in terms of biomass and yield to its near isogenic line M37W, and that carotenoid production should be stable under a variety of conditions and environments.8) without fertilizer treatment (N0), the LAI of Carolight® plants was significantly higher than that of M37W plants, but by silking (R1) no significant differences were detected between the genotypes. The early differences in LAI between genotypes were probably not substantial enough to cause statistically significant differences in radiation interception, the rate of photosynthesis or the duration of photosynthesis (11During early growth (Vynthesis . Furtherynthesis . These r200) most of the measured traits remained at similar levels compared to the N0 treatment  are limited in cereals, and have been reported mainly in other crops such as tomato363839et al.Capsicum annuum and C. chinense produced more carotenoids than field-grown plants, reflecting the 20% higher light intensity in the field compared to the greenhouseC. annuum cultivars has also been shown to decline by up to three-fold in the field compared to the greenhouseWe observed a 25% higher total carotenoid content in the young endosperm of greenhouse plants compared to field plants, but this gap had closed by maturity (60 DAP) with plants in both environments accumulating ~95 μg gPacrtI (phytoene desaturase), which converts phytoene into lycopene. This finding is important because it suggests that the performance of PacrtI might not be optimal, and this could be remedied in subsequent Carolight® products, particularly with the rapid development of genome editing technologies421PacrtI mRNA, and whether the transient differences in β-branch carotenoids also reflected differences at the mRNA level. In both environments and at all time points, PacrtI mRNA accumulated in the endosperm at significantly lower levels (~3-fold lower) than Zmpsy1 mRNA. We also found that greenhouse plants accumulated twice as much Zmpsy1 mRNA in the young endosperm (15 DAP) than field plants which partially explains the transient differences in carotenoid profiles we observed. This is consistent with the significant difference in the total carotenoid content of the endosperm of greenhouse and field plants at 25 DAP, suggesting that Zmpsy1 mRNA accumulates at higher levels in the endosperm of greenhouse plants at ~15 DAP thus increasing the flux in the downstream carotenoid pathway. Zmpsy1 mRNA levels then converged in the greenhouse and field plants between 20 and 30 DAP. Similarly, a correlation between psy1 expression and total carotenoid levels was specifically observed at 20 DAPZmpsy1 mRNA alone while other endogenous or exogenous carotenogenic transcripts were expressed at consistent levels suggests that Zmpsy1 mRNA and/or its promoter is peculiarly sensitive to the different environmental conditions in the greenhouse and field. These environments are characterized by differences in light quality and temperature, as well as biotic and abiotic stresses, and this could influence carotenogenic gene/transgene expression or carotenoid accumulation. Light regulates genes and gene products related to photosynthesis, including carotenoidsOspsy1 and Ospsy2 contain cis-acting elements involved in light regulationZmpsy1-348psy genes in corn have overlapping functions in the modulation of carotenoid synthesis in different tissues and in response to developmental and/or stress signals. In corn leaves, carotenogenesis may require both phytochrome-dependent and phytochrome-independent photoregulation of psy2 as well as non-photoregulation of psy151Zmpsy2 mRNA levels in the leaves and Zmpsy3 mRNA levels in the roots, Zmpsy1 mRNA levels do not appear to respond to abiotic stress at all. Importantly, these studies were performed under laboratory conditions and there are no studies thus far reporting the response of Zmpsy1 to the combined stresses that may be encountered under field conditions. Interestingly, we controlled the Zmpsy1 transgene using the wheat low-molecular-weight glutenin promoter, which is not known to be influenced by light or abiotic stress. Our study therefore provides the first evidence that environmental factors can influence either this promoter or the Zmpsy1 gene.When we analyzed the carotenoid profile, the proportions of the major carotenoids found in the endosperm varied during development between the plants in the greenhouse and field, although again these differences had disappeared by maturity and the profiles in mature kernels were similar regardless of where the plants were cultivated. The young endosperm tissue of field plants contained ~20% higher levels of β-branch carotenoids than greenhouse plants, but this reflected the specific accumulation of more zeaxanthin, β-cryptoxanthin and antheraxanthin and not β-carotene. Indeed, β-carotene was only ~3% more abundant in the greenhouse plants, consistent with the negative response of β-carotene to light intensity in tomatoZmlycb mRNA was four times more abundant than Zmlyce mRNA in both environments, which is important because the corresponding enzymes compete to divert flux into the β-branch and ε-branch545657Zmbch1 also promotes the accumulation of α-carotene55Porphyra umbilicalis, is also responsible for β-ring hydroxylationZmbch2 mRNA accumulated to levels 100-fold greater than Zmbch1 mRNA and that Zmcyp97a and Zmcyp97b were expressed at levels at least 10-fold higher than Zmcyp97c in the endosperm of both the greenhouse and field plants. The higher content of β-branch carotenoids in the Carolight® endosperm in both environments can therefore be explained by these specific gene expression profiles.Although we did not observe any major differences between greenhouse and field plants in the expression of most endogenous carotenogenic genes during endosperm development (15–30 DAP), Zmpsy1) thus showing that even relatively minor differences in the regulation of transgene expression can affect the behavior of engineered crop plants in a significant and environmental-dependent manner.In conclusion, we have shown that the high-carotenoid transgenic corn line Carolight® does not behave in a significantly different manner, in terms of phenology and agronomic performance, to its wild-type counterpart. Importantly, we tested for differences under diverse environmental conditions to confirm that the engineered variety is likely to perform well in a range of environments relevant to commercial deployment. To our knowledge, this is the first time that a transgenic variety with a second-generation output trait has been characterized in such a comprehensive manner although such comparisons are highly recommended during the development of novel crops to avoid disappointing field performance and late-stage product development failure. Minor differences in parameters such as endosperm carotenoid composition and LAI were measured in the early stages of development, but they did not affect the grain yield or any other growth/development phenotypes. Importantly we provide a molecular basis for this behavior which might help the development of improved versions not only of Carolight® but also other second-generation and third-generation crops. Carolight® also behaves in a similar manner in a controlled environment and under field conditions, which is an essential requirement to support further product development given that early studies are restricted to the greenhouse under the current regulatory framework and field studies are only permitted with permission from national competent bodies. The transient minor differences we observed appear to reflect differences in the expression of a single gene .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We manipulate and verify the redox state of single metalloprotein crystals by combining electrochemical control with synchrotron infrared microspectroscopy. Escherichia coli we demonstrate fully reversible and uniform electrochemical reduction from the oxidised inactive to the fully reduced state, and temporally resolve steps during this reduction.We describe an approach to generating and verifying well-defined redox states in metalloprotein single crystals by combining electrochemical control with synchrotron infrared microspectroscopic imaging. For NiFe hydrogenase 1 from  In this way, a number of redox levels of the NiFe site have been characterised by applying IR spectroscopy or spectroelectrochemistry to the enzymes in solution,a–SI with a vacant bridging site that becomes occupied by a bridging hydride upon proton-coupled reduction to the Nia–C or Nia–R levels.a–SI is also the state which reacts with H2 to initiate the H2 oxidation catalytic cycle.a–C is in tautomeric equilibrium with Nia–L, in which the hydride has moved as a proton to a nearby basic site. Nia–R and Nia–L each represent a collection of up to three sub-states which are thought to differ in protonation close to the metal centre.a–R sub-states, which otherwise appear simultaneously in solution.We demonstrate this approach using single crystals of NiFe hydrogenase 1 (Hyd1) from E) were converted to volts (V) vs. the standard hydrogen electrode (SHE) using the conversion E(SHE) = E(SCE) + 241 mV.A number of groups have reported IR microspectroscopy cells for measurements under electrochemical control.2 window  was then placed over the crystal and the cell was sealed using two PTFE gaskets . Microspectroscopy experiments were carried out on the MIRIAM beamline at the Diamond Light Source, UK, using a Vertex 80V FT-IR spectrometer and a Hyperion 3000 IR microscope (Bruker) with a high-sensitivity photovoltaic mercury cadmium telluride (MCT) detector cooled to 77 K with liquid N2. Spectra were acquired in reflection mode  using a 36× objective and 15 × 15 μm2 aperture, as an average of 1024 interferograms at 4 cm–1 resolution and 40 kHz interferometer frequency. Data acquisition was performed using Bruker Optik's OPUS software (version 7.0), and data analysis was carried out using OriginPro software (OriginLab). Electrochemical control was achieved using an Autolab 128N potentiostat (EcoChemie) controlled by Nova software (version 1.10). A representative cyclic voltammogram of the crystallisation buffer containing redox mediators is shown in Fig. S1 of the ESI.Crystals of Hyd1 were manually harvested using a cryo-loop (Hampton Research) and transferred into a 5–10 μL droplet of crystallisation buffer on the working electrode with the aid of an optical microscope see ESI. An addi2 in cross section and 1000–2000 μm in length FeII, –1, Nia–RII and Nia–RIII, previously characterised in Hyd1,–1, presumably corresponding to several Nia–L states.Visible images recorded of the crystal over a period of several hours show no noticeable dissolution or change in dimension throughout the measurement  that contributed to the results presented here. We also thank Elena Nomerotskaia for assistance with isolation of Hyd1.Supplementary informationClick here for additional data file.


MDA-MB-231 tumor bearing mice were treated either with high (46 °C) or low dose (42 °C) water hyperthermia for 60 min. Changes of GRPR and αvβ3 integrin expression were assessed via multiplexed optical imaging. Vascularization was reconstructed and quantified by µCT imaging after contrast agent injection. We found that high dose hyperthermia is capable of increasing the expression of GRPR, αvβ3 integrin, CD31, and Ki67 in tumors. Also the morphology of tumor vasculature changed . Low dose hyperthermia induced comparatively moderate effects on the investigated protein expression pattern and vascular remodeling. We conclude that under defined circumstances, specific temperature doses affect the reorganization of tumor regrowth, which is triggered by residual “dormant” cells even though tumor volumes are transiently decreasing. Further on, GRPR, αvβ3 integrin expression are versatile tools to surveil potential tumor regrow during therapy, beyond the conventional determination of tumor volumes.The outcome of tumor treatment via hyperthermia in the clinic has been reported to be heterogeneous. Here, we assessed how the presence of gastrin-releasing peptide receptor (GRPR) and α Examples are the sensitization of cells to ionizing radiation or chemotherapeutic agents via hyperthermia4, the hyperthermic perfusion combined with surgery or chemotherapy in case of peritoneal metastases5, and a general sensitization of tumor cells for subsequent chemotherapy sessions. As a single therapeutic modality the therapeutic outcome of hyperthermia was reported to be quite heterogeneous6, particularly when external heating sources  are used.Hyperthermia has been recognized as a potent therapeutic strategy against cancer. Despite of it, its transfer and implementation into the clinical situation progresses slowly. Namely, the current clinical practice confines hyperthermia as a supportive modality, which is used in combination with other therapy regimesTo the best of our knowledge, there is no consistent understanding of the biomedical reasons which are responsible for the heterogeneity of the therapy success of hyperthermia. Such investigations will require the utilization of specific protein structures, which allow the monitoring of the impact of hyperthermia on the target tumor tissue.8, prostate9, lung carcinoma11 breast cancer12, etc. GRPR has been extensively targeted with radiotracers in the preclinical and the preclinical level using bombesin 13 analogues in radioactive or optical probes15. Its widespread presence in many tumors and its intensive use in imaging approaches on the molecular level make the GRPR a very attractive marker for monitoring of the tumor therapeutic efficiency of hyperthermia.Importantly, the gastrin-releasing peptide receptor (GRPR) is expressed in multiple cancers such as colonvβ3 integrin. In particular, in whole body imaging applications αvβ3 integrin is capable of assessing the presence of tumor vascularity18, particularly in post-therapy monitoring applications19. Namely, αvβ3 integrin is present in activated endothelial cells of newly formed vasculature in tumors. The presence of vascularity is essential for tumor growth and metastasis. For this reason, αvβ3 integrin is an appealing marker for monitoring the effects of hyperthermia on vascularity.A further molecular marker which has also been widely recognized to be expressed in tumors is αvβ3 integrin after hyperthermia treatment in vivo is not known so far. Therefore, we sought to address GRPR and αvβ3 integrin in tumors in a multiplexed whole body-imaging approach in laboratory animals in order to evaluate the effect of hyperthermia at different temperatures and asked the following questions: First, which effects do different temperatures exert on GRPR expression and cell viability? Second, how does high or low dose hyperthermia influence the expression of αvβ3 integrin and GRPR in a breast cancer xenograft? Third, to what extent does hyperthermia alter the intratumoral vessel density and relative blood volume? The knowledge of these relationships would allow a better understanding of the underlying biomedical processes and consequently a refinement of the tumor therapeutic strategies.In general, the particular behavior of GRPR and αFlow cytometry analysis showed that GRPR expression was enhanced in cells treated with hyperthermia at 44 °C (24 h post therapy) and increased even more when temperatures increased to 46 °C (high dose hyperthermia)  of fluorescence intensities in tumors after administration either of a GRPR or an αvβ3 integrin probe in tumors treated with high dose hyperthermia  compared to the non-treated control ones  accumulated mostly in the tumor and kidney . Interestingly, a distinct fluorescence was also seen in the kidney of untreated mice Fig. . In geneEx vivo µCT imaging showed that the relative blood volume (rBV) in the tumor and its periphery, as an indicator for the vascularization grade, increased distinctly after high dose hyperthermia  high dose hyperthermia in vivo reduces tumor volumes and concomitantly increases GRPR, αvβ3 integrin, CD31, and Ki67 in these tumors, (3) high dose hyperthermia also changes the morphology of tumor vasculature by increasing the relative blood volume (rBV), the small-diameter vessel density, and by decreasing the expression of α-SMA, an indicator of the endothelial cell maturity in those tumors, (4) low dose hyperthermia  induces only moderate effects .In this study we were able to show that (1) high dose hyperthermia  is capable of inducing both apoptosis and the expression of GRPR and α20. This means that a distinct heat stress has been induced particularly with the high temperature dose protocol .Interestingly, GRPR expression in isolated tumor cells was particularly prominent in those cell populations which were exposed to comparatively high temperature doses. The effect was accompanied by enhanced caspase-3 activation (cleaved caspase-3) which is a marker of irreversible cell damage and also for apoptosisin vivo situation in mice. Fluorescence signals of the GRPR were visible in tumors particularly at 8 days post treatment. These cells were able to express the proliferation marker Ki67, as immunohistochemistry analysis of tissues with high fluorescence revealed. Therefore, we assume that cells which managed to survive a strong heating stimulus are able to induce protein expression as response to thermal stress (see below).By applying a heating stimulus, more or less viable residual cells were still able to actively induce protein expression. The increased presence of GRPR after hyperthermia treatment was also observed in the vβ3 integrin expression in tumors, particularly after high dose hyperthermia. Therefore, these molecular structures might well be metabolically linked between each other as response to a heating stress. Namely, the impairment of the tumor vascularity due to hyperthermia leads to hypoxia, as a result of impaired nutrient supply. Hypoxia triggers HIF-122, which in turn promotes the expression of VEGF-124 as stress response reaction. VEGF was recognized to induce the expression and activation of EGFR26 and GRPR26. Additionally, the activation of one of these receptors might affect the activation of other ones30. Such mechanisms could be one reason for the enhanced proliferation (as seen by the expression of Ki67 in tumor tissue samples) and increased presence of GRPR in residual tumor tissue after hyperthermia.The increase of GRPR was accompanied by an increase of α31 after the hyperthermic stress (see above). Our observations evidence that blood vessels are newly formed in treated tumors .High dose hyperthermia seems to change the morphology of tumor vasculature as revealed by a high relative blood vessel volume and an increased blood vessel density compared to non-treated tumors. These observations are, again, signs for the induction of pro-angiogenic processes, as a result of the release of pro-angiogenic factors from residual tumor cells32. Autophagy, a process of cellular consumption, is thought to represent an energy conservation effort of cells which managed to escape from cell death. We postulate that heating can efficiently kill tumor cells but it might also leave some residual cells undergoing autophagy. They represent “dormant” cells in the tumor capable of recovering when the growth conditions have been restored31. The potential of recovery is underlined by the presence of protein expression (GRPR) and changed vascular morphology discussed above, together with the presence of cells expressing the proliferation marker Ki67. Such a response pattern is the manifestation of hormesis34. As a result of hormesis, spreading of tumor cells with ongoing time is favored, particularly if one does not undertake any additional therapeutic treatments. Namely, it is known that an increased vascular density36, hypoxia37 and a low expression of α-SMA39 is beneficial for tumor cells to regrowth and metastasize. The same applies for the increased blood volume in tumors in conjunction with mitogenic and morphogenic effects of GRPR42, and finally the potential of GRPR to co-activate EGFR30 and VEGF29.Irrespective of the encountered morphological features of tumor vascularity and protein expression, a reduction of tumor volumes was found after high dose hyperthermia (at least at 8 days post treatment). We associate such effects with loss of cells due to apoptosis and shrinkage of cells undergoing autophagyvβ3 integrin expression in tumors were observed at 8 h post hyperthermia is related to the time the dormant tumor cells (in autophagy) need to trigger the pathophysiological pathways for metabolic restoration in the sense of “hormesis”. In particular, dormant cells “recognizing” hypoxic conditions have to induce the expression of relevant molecules such as VEGF-1, EGFR, GRPR, etc. (see above). This is a time-consuming process, which implicates the expression of new mRNA and protein molecules.The fact that the effects on GRPR and αvβ3 integrin, CD31, Ki67, and α-SMA expression were less prominent in the low dose hyperthermia group. Apparently, the applied heating stress was too low to induce remarkable heating responses. The fact that the tumor volume suddenly increased after 24 h after treatment might be ascribed to the induction of edema43 as a result of the permeabilization of the tumor vasculature without any morphological impairment. All these effects might represent a “mild” response to hyperthermia, by which less (or no) cells undergo autophagy and hormesis.Interestingly, GRPR, αvβ3 integrin to hormesis. Accordingly, we suggest the following strategy for hyperthermic treatments of tumors: a) particularly when using high dose hyperthermia, physicians should imperatively consider repeated and complementary therapy sessions together with an appropriate image-based therapeutic surveillance of recovery signs in short times intervals after therapy, b) low dose hyperthermia is attractive because it does not trigger “hormesis” as much as high dose hyperthermia. Hereto, repeated “mild” therapy sessions can stress cells for additional therapeutic modalities  and should be applied together with a close-meshed image-based therapeutic monitoring. It might lead to smooth tumor eradications. In this context, changes of GRPR and αvβ3 integrin expression could be used to identify a potential reorganization of tumor regrowth.All in all, we attribute the enhanced expression of GRPR and αvβ3 integrin optical probe accumulation in the kidneys. We postulate that the release of cell debris from the tumor to the blood as a result of the hyperthermic tumor treatment might lead to a systemic release of cytokines . It is widely known that cytokines reduce glomerular filtration rate but they can also be beneficial in ameliorating the host immune response against cancer cells, which has repeatedly been claimed in the literature44. Another, quite simple, explanation is clearance of probe metabolites, an issue which might be further clarified in future investigations.Interestingly, the hyperthermic treatment of tumors reduced the GRPR and α45. Its inhibition might also increase the effect of hyperthermia in analogy as it has already been with chemotherapy46. Furthermore, αvβ3 integrin is known to play an important role in adhesion of newly formed endothelial cells in the tumor extracellular matrix. Its inhibition might reduce migration of endothelial cells47, and prevent proliferative kinase cross talks (e.g. with the extracellular regulated kinase (ERK1/2)48.The increased expression of GRPR and αVβ3 integrin might be beneficial for certain combination therapies targeting these proteins additionally to hyperthermia. For example, a GRPR inhibitor might have an effect on tumor cell proliferation, since GRPR stimulates tumor growthVβ3 integrin expression might be used to assess reorganization of tumors as consequence of an adaptive response (hormesis) of dormant (authopagic) cells to the heat stress stimulus. Such effects have to be considered more in detail in future. In this sense, we shed more light into the question why tumors treated at temperatures above the widely recognized thresholds previously established from in vitro experiments50 are still able to proliferate. In the in vivo situation, the response to a heating stimulus has a higher degree of metabolic and physiological complexity, as discussed above. In this context, hyperthermia is a subject of molecular and personalized medicine, as it has been claimed for other oncological modalities as well.In this study we gave first evidence that GRPR and αvβ3 integrin, the relative blood volume (rBV), the small-diameter vessel density, and decreased expression of α-SMA. The strong response was suggested to be associated with “dormant” residual cells in the tumor region capable of organizing recovery. By utilization of lower treatment temperatures, these effects were less prominent. This distinct response pattern might well be an explanation why the therapeutic outcome of hyperthermia in the clinic has been so heterogeneous until now. Consequently, we propose to consider: (a) repeated high dose hyperthermia together with complementary therapy sessions or (b) to use repeated “mild” hyperthermia sessions in order to stress tumor cells for additional therapeutic modalities  and c) to also reconsider the presently established thresholds for temperature dosages and therapeutic strategies when treating tumors via hyperthermia as an adjunct to current oncologic modalities. This means that the parameters GRPR, αvβ3 integrin expression, and changed vascular morphology do reflect potential regrowth features of tumors after hyperthermia treatment. They might be exploited for an image-based therapeutic monitoring of hyperthermia in the future.We have shown that high dose hyperthermia induces a strong response of the tumor tissue in terms of an overexpression of GRPR and changed vascular morphology; it is a process, which we identified by the presence of α2 atmosphere. In a previous experiment, the cells were shown to be able to express GRPR  was cultured in DMEM Ham’s F12 (1:1) (Gibco) containing 5% FCS (Gibco) at 37 °C in a humidified 5% CO2, humidified air).To differentiate temperatures having only transient or lethal cytotoxic effects, cells were seeded 24 h prior to treatment in a culture flask. To exactly measure the temperature during hyperthermia , a fiber-optic thermocouple (OPTOCON AG) was used. The duration of treatment was 1 h. Immediately after hyperthermia, cells were incubated at standard conditions. An untreated control maintained at standard culture conditions  hyperthermia, cells were harvested and washed in ice-cold phosphate buffered saline (PBS). The cells were fixed in 2% formaldehyde and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). The GRPR and cleaved caspase-3 were stained indirectly using a FITC-conjugated anti-rabbit IgG secondary antibody (abcam). The following primary antibodies were used: The rabbit-anti-GRPR antibody (antibodies-online Inc.) and the monoclonal rabbit-anti-cleaved caspase-3 antibody . The measured values were normalized to untreated control cells, seeded, harvested, and stained at the same point of time. Isotype, negative, and positive controls were used  and phosphatase inhibitor cocktail (Roche Diagnostics). Protein concentration was determined using the Bradford assay. Protein loads of 5 µg were resolved by SDS-PAGE and analyzed by immunoblotting. The following primary antibodies were used: anti-GRPR antibody (antibodies-online) and anti-α-tubulin antibody (Boehring). The second antibody was a polyclonal HRP labelled goat-anti-rabbit IgG (Santa Cruz Biotechnology). Isotype, negative, and positive controls were used  and implanted subcutaneously in the middle of the back of the animals.All experiments were in accordance with international guidelines on the ethical use of animals and were approved by the regional animal care committee (No. 02-068/11). During experimentation, the animals were anesthetized with 2.0–2.5% Isoflurane (Actavis). For tumor implantation, 2 × 10In vivo external hyperthermia treatments were performed via exposure of tumors to hot water (n = 8). During low dose hyperthermia, the tumor surface exhibited a temperature of 42 °C, and a minimum of 40 °C in the inner tumor tissue; during high dose hyperthermia, temperatures of 46 °C were reached at the tumor surface and at least 44 °C in the inner tumor area. The hyperthermia treatment was conducted for 1 h. The tumor and body temperatures were monitored using fiber-optic thermocouples (OPTOCON AG). These preconditions were determined in a prototype experiment. Tumor size was measured 24 h, 48 h, and 8 days after hot water hyperthermia. The tumor volume was calculated by the formula V = π/6 × length × width × height of the tumor51.® 800CW RGD and 2 nmol BombesinRSense™ 680 per mouse was done simultaneously at 24 h and 7 days after treatment in order to image change of tumor vascularization, probe accessibility after treatment and GRPR expression. 24 h after administration of the optical probes, the fluorescence intensities were monitored by whole body near infrared fluorescence (NIRF) imaging system (Cri Inc.) using the filter “Deep Red”  for IRDye® 800CW RGD, and the filter “Yellow”  for BombesinRSense™ 680. Comparatibility of fluorescence intensity of each image was attained by normalization to scaled counts per second. Each contrast agent has been analyzed separately for its fluorescence spectrum after subtracting the spectrum of autofluorescence. The contrast of each contrast agent was determined through the tumor-to-muscle-ratio (TMR), i.e. by dividing the average fluorescence signal of a defined region of interest (ROI) in the tumor area and the fluorescence signal of normal tissue . The TMR for each optical probe and the point of time after hyperthermia were related to the native images (without contrast agent) in order to exclude natural intrinsic contrast at the wavelengths of interest. Contrast agent accumulation was compared to the non-treated control group (n = 10).The intravenous injection of 1 nmol IRDyein vivo optical imaging.At 8 days after treatment, the fluorescence intensity of dissected organs was measured under the same instrument settings as described for et al.52. In this context, the mice were perfused with 10 ml of the lead-based silicon rubber casting resin Microfil® MV-130 and MV-diluent, prepared according to the manufactor’s guidelines (Flow Tech). All scans were performed using a dual source µCT cone-beam scanner TomoScope® Synergy (CT Imaging). Scans were visualized and analyzed using Imalytics® preclinical software (Philips Research) after three-dimensional volume rendering of reconstructed high-resolution µCT data sets. In order to analyze the tumor-associated vessels located in the immediate periphery of the tumor, we defined three consecutive areas over the tumor surface, whereas each coat has a defined width of 0.592 mm (8 voxel). The total surface area of analysis was of 1.776 mm (24 voxel) . The following antibodies were used: For detection of Ki67 primary antibody was a monoclonal anti-rabbit-Ki67 antibody (abcam) and isotype control a monoclonal rabbit IgG (abcam) (primary antibodies). For detection of GRPR: monoclonal rabbit-anti-human-GRPR-antibody (antibodies-online Inc.), a polyclonal rabbit IgG (h & l) antibody (Thermo Scientific) (isotype control). CD31: polyclonal rabbit anti-CD31 antibody (abcam), rabbit polyclonal IgG (abcam) as isotype control. α-SMA: monoclonal mouse anti α-SMA (abcam) as primary antibody and Mouse IgG2a (BIOZOL Diagnostica) as isotype control. The detection of the markers was performed either with addition of streptavidin-alkaline phosphatase or peroxidase labelled system (both from Dako). For tissue counterstaining, nuclear structures were stained with Mayer’s Hematoxylin (Fluka/Sigma-Aldrich). Quantification was performed using the software cellSens (Olympus). Positive, isotype, and negative controls of immunostaining were used (Supplementary Figure Data are expressed as means ± SEM or standard deviation respectively. Statistical significance was analyzed by Mann-Whitney. A p-value of 0.05 or less was considered statistically significant. The number of parallels or animals in the experiments is given in the figure legends.Supplementary Information