This retains the possibility of using low‐power rectangular pulses for 1H‐decoupled 3D 31P MRSI  but increases the SNR with the receive sensitivity of 31P surface coils. The performance of the combined coil setup was evaluated by measuring 1H and 31P SNR with and without the 31P receive array and by assessing the effect of the receive array on the transmit efficiencies of the birdcage coil.We developed an 8‐channel 31P insert in combination with the birdcage achieved an average 31P SNR gain of 1.4 ± 0.4 in a center partition of the brain. The insert did not cause losses in 1H MRS performance and transmit efficiency, whereas for 31P approximately 20% more power was needed to achieve the same γB1.Compared to the birdcage coil alone, the 1H MRSI and optimal 1H‐decoupled 3D 31P MRSI, with increased SNR of the human brain without patient repositioning, for clinical and research purposes at 3 Tesla.The new coil configuration allows  It is used to study several neurological diseases and other pathologies, such as cancer, in which metabolite levels are altered.In vivo phosphorus MRS 3P MRS of 31P MRS in the noninvasive examination of brain diseases has already been demonstrated for this purpose many years ago , it is much less used than 1H MRS,31P MR spectra of the human brain, it is common to employ a birdcage type of coil for transmit/receive with either a single or multivoxel MRSI pulse sequence.0‐shimming, it is required that the 31P coil is integrated with a 1H radiofrequency (RF) element, which can also be used for 1H‐decoupling, inducing the nuclear Overhauser effect and 1H MRS.Although the unique value of 31P MRS compared to 1H MRS can be compensated by prolonging acquisition times, enlarging voxels, and/or using a higher magnetic field. However, sensitivity can also be enhanced by reducing the size of the receiving RF coils, in particular at the more superficial regions of the brain.31P experiments to apply RF‐pulses for 1H‐decoupling and/or inducing nuclear Overhauser effect for increased spectral resolution and SNR, respectively.To overcome that low SNR hampers quantification of metabolite peaks, the lower sensitivity of 31P phased array coil insert to be used in combination with a commercially available 1H/31P birdcage coil has not been designed for the brain at 3T.Because 3 Tesla (T) systems are widely available in the clinic, many sites could take advantage of an improved coil design rather than switching to expensive ultrahigh field (≥7T) systems. Although X‐nuclei phased array coils are not new,31P MRS at 3T, by developing a dedicated 8‐channel 31P receive head‐array coil that can be used in combination with a 1H/31P birdcage transmit‐receive coil to enable 1H‐decoupling and nuclear Overhauser effect. We acquired in vivo 31P spectra of the brain and compared the performance of the coil setup with and without the 31P 8‐channel insert.In this work, we aimed to maximize SNR and spectral resolution, and thereby the applicability of 22.131P receive head‐array coil was designed to be combined with a commercially available quadrature Tx/Rx 1H/31P birdcage coil . Both coils were actively detunable at the 31P frequency. To this end, the commercial coil had to be adjusted by the manufacturer. For 1H applications, the birdcage coil was always used to both transmit and receive.An 8‐channel The elements of the head array were constructed using 12.7 mm‐wide copper tape with a thickness of 35.6 µm on a Plexiglas cylinder with an outer diameter of 24.5 cm. The dimensions of the elements were 100 × 200 mm, except for the frontal element (100 × 100 mm) to create space for the patient’s nose and view, herewith improving patient comfort  containing phosphoric acid. Temperature measurements were performed with an optical probe  positioned in a small gel phantom with 3 wt% agar and 0.5 wt% NaCl while transmitting at maximum RF power of either 31P transmit efficiencies were compared by means of a slice selective pulse‐acquire experiment (TR = 15 s) covering the brain through assessment of the voltage that corresponds to a γB1 of 500 Hz, reflected by a maximized PCr magnitude signal. 1H transmit efficiencies were determined automatically by the MR system. To determine the noise correlation between the array elements, a single‐slice 31P gradient‐echo image was acquired while transmit power was set to 0.The performance of the birdcage alone and the combined setup were tested with 3 healthy volunteers  on a 3T MR‐system . The 1H‐decoupling scheme was used to acquire 31P MR spectroscopic images of the whole brain for SNR comparisons between both coil setups. TR was 2000 ms. For excitation, a pulse with a duration of 500 µs and a flip angle of 40°  were applied. The dead time between pulse and acquisition was 100 μs. For 3D 31P MRSI, the FOV was set to 260 × 260 × 260 mm3 applying Hamming‐weighted k‐space sampling, averaging 4 FIDs of 1024 data points around the center of a 10 × 10 × 10 k‐space matrix. The voxel size defined as 64% of the point spread function area was approximately 40 cm3.1H‐decoupling (γB1 = 250 Hz) was turned on during the first 256 ms (50%) of the acquisition window.A 3D MRSI FID sequence with a WALTZ4 (Wideband Alternating phase Low‐power Technique for Zero residue splitting) 1H SNR comparison, 1H MR spectra of the anterior part of the occipital lobe were acquired with a single voxel PRESS sequence using chemical shift selective water suppression.3 voxel with a flip angle of 90° (64 averages).For All volunteer studies were conducted with approval of the institutional review board of the Radboud university medical center, The Netherlands.2.4The amplitude and relative phase of the signal of each array coil element depend on the magnetization and position of the excited volume with respect to the receive elements. Because these parameters vary per array coil element, we used the whitened singular value decomposition algorithm for signal combination.31P MRSI data set was interpolated to a 16 × 16 × 16 matrix with a nominal voxel size of 16.25 × 16.25 × 16.25 mm3. The SNR performance of the birdcage alone and the combination with the phased‐array insert were evaluated in the center transversal partition of the 3D dataset in all volunteers. SNR was calculated as the ratio of the integral of the PCr peak, as fitted by Metabolite Report in syngo , divided by the SD of the noise. Noise SD was calculated from a signal‐free portion of the spectrum. An average noise correlation matrix was calculated from the noise in the 31P gradient‐echo images.Before Fourier transform, the in vivo 3D 1H performance of the birdcage with and without the 31P array inserted, the SNRs of all single voxel 1H spectra, acquired from the anterior part of the occipital lobe, were analyzed with LCModel version 6.3‐0C software using a simulated basis set of 24 metabolites.To evaluate the 331P receive head array, we determined the Q‐factor ratio QUL/QL for all individual array elements. The values for the Q ratio ranged from 1.4 to 2, depending on the head‐to‐element distance. Next, we determined the port‐to‐port (S21) decoupling, which ranged from −10 dB to −15 dB between neighboring elements and from −8.5 dB to −22.6 dB for non‐neighboring elements. The calculated noise correlation matrix showed variations in noise correlation between both neighboring and non‐neighboring elements  and PCr signals, respectively, was studied with the 1H/31P birdcage coil only and with a combination of this coil and the 31P receive head‐array insert.The presence of the array coil did not cause any losses in 1H/31P birdcage coil has a flat 31P receive profile, and the combined probe showed a 31P profile with a radially increasing sensitivity from the center of the head toward the array elements. The SNR gain of the PCr peak due to the insertion of the 31P array coil varied from 0.9 in the center up to a factor of 3.2 in the anterior part of the brain. The average SNR gain (± SD) of all selected voxels was 1.4 ± 0.4 (Figure The 1H MRS sensitivity for both coil configurations, we used the SNR value of the NAA methyl peak at 2.01 ppm. For the birdcage, the 1H SNR of a volume in the occipital lobe of the brain was 19, 20, and 17 for volunteers 1, 2, and 3, respectively. After inserting the 31P head array, the SNRs were 20, 18, and 19. Ergo, the SNRs of 1H MR spectra were not influenced by the presence of the 31P array coil.To assess the 1H‐31P J‐coupling. Applying 1H‐decoupling can remove this heteronuclear coupling and results in well‐resolved signals of phosphoethanolamine, phosphocholine, glycerophosphoethanolamine, glycerophosphocholine, and adenosine triphosphate. 31P signals were not affected by spurious signals or additional noise due to 1H‐decoupling (Figure With a standard FID MRSI sequence, the resonances of phosphomonoesters, phosphodiesters, and adenosine triphosphate are not well resolved due to g Figure .431P 8‐channel receive‐array insert with a double‐tuned 1H/31P birdcage transmit/receive coil to enable single‐session combined 31P and 1H MRSI examinations without the need of repositioning the patient in clinical routine, trials, and research at 3T.In this work, we combined a home‐built 31P MR spectroscopy and spectroscopic imaging mostly have been performed with surface coils. Because RF transmit surface coils have an extremely inhomogeneous transmit field, the flip angles of conventional excitation pulses depend on the distance to the coil and need calibration. This can be largely overcome by adiabatic pulses.1H‐decoupling and 1H‐31P nuclear Overhauser effect are applied. Our combined coil configuration takes advantage of separated transmit and receive elements. This allows us to generate a homogeneous transmit field with the volume 1H‐31P birdcage coil and to achieve a high sensitivity close to the array coil. For excitation, this allows us to employ rectangular pulses at lower RF power. Because shorter repetition times can be achieved with rectangular pulses, flip angle calibration can be performed fast and more averages with small flip angles acquired in the same amount of time, resulting in a higher SNR per unit time.Traditionally, With the head‐array coil insert, we achieved an increase up to 3.2‐fold in SNR in superficial anterior brain areas compared to the birdcage coil alone while almost maintaining similar SNR in the center of the brain. This SNR profile is in line with other studies using an array of coils.1H coil31P and 1H home‐built coils: 0.1UL/QL ratio of an unloaded single element is approximately halved when placed inside the birdcage. The ratio also depends on the element position in the birdcage, which implies a shared mutual resistance. Furthermore, with both the 8‐channel array and a sample present inside the birdcage, location‐dependent coupling caused extra interelement coupling of the array elements. A better decoupling between birdcage and receive elements would further improve the performance of the array. However, for safety and regulatory reasons it was not possible to make modifications to the commercial coil.Noise correlation values of the array coil ranged from 0.05 up to 0.66, with a mean of 0.31 ± 0.16. This is comparable to reported values of a commercially available 8‐channel 1 for 31P of the birdcage was reduced by ~20% in the presence of the array coil, the probe was still capable of generating a 90° flip angle with a bandwidth of approximately 48 ppm, which is sufficient to excite all 31P spins relevant for in vivo applications.Although the maximum γB531P head array with a double‐tuned 31P/1H birdcage offers the advantages of an increase in 31P SNR while retaining a homogeneous transmit field on both frequencies. The losses in 1H performance and the 31P‐transmit performance of the birdcage were negligible or could be overcome. Our setup facilitates combined in vivo 1H and 31P MRSI examinations on a clinical 3T system without repositioning the subject. The proposed coil configuration in this study paves the way for human applications at 3T with more advanced sequences such as selectively refocused insensitive nuclei enhanced by polarization transfer.31P and 1H MRS acquisitions on current state‐of‐the‐art MR‐systems.The combination of an 8‐channel


This study demonstrates magnetization transfer (MT) effects directly affect relaxometry measurements and develops a framework that allows single‐pool models to be valid in 2‐pool MT systems.B1rms{\text{B}}_{1}^{{{\text{rms}}}}) is kept fixed across all measurements. A practical method for achieving controlled saturation magnetization transfer (CSMT) using multiband RF pulses is proposed. Numerical, Phantom, and in vivo validations were performed directly comparing steady state (SS) estimation approaches that under correct single‐pool assumptions would be expected to vary in precision but not accuracy.A theoretical framework is developed in which a 2‐pool MT system effectively behaves as a single‐pool if the RMS RF magnetic field  and TRs are used if the CSMT approach is not used. Using CSMT, stable measurements of both T1 and T2 are obtained. The measured T1(T1CSMT)) depends on B1rms{\text{B}}_{1}^{{{\text{rms}}}}, which is therefore an important parameter to specify.Numerical simulations predict single‐pool estimates obtained from MT model generated data are not consistent for different SS estimation methods, and a systematic underestimation of TThis work demonstrates that conventional single pool relaxometry, which is highly efficient for human studies, results in unreliable parameter estimates in biological tissues because of MT effects. The proposed CSMT framework is shown to allow single‐pool assumptions to be valid, enabling reliable and efficient quantitative imaging to be performed. Extensive literature has explored interaction between these pools, which is often referred to as magnetization transfer (MT).MRI is routinely used as a highly sensitive soft‐tissue imaging modality invaluable for clinical diagnosis. MR images are qualitative, in that the voxel intensity values are related to underlying tissue properties, but are also dependent on specific details of scanner hardware and softwareM0 or proton density, T1 and T2.1/2 (DESPOT),1 values for healthy white matter (WM) at 3 T range from 699‐1735 ms, which is much larger than the estimated parameter uncertainty for individual studies. T2 values measured by these methods are also routinely reported as lower than expected.0 and T1 definitions to be defined by MT rather than being hindered by it. We achieve this by observing that for short TR steady state (SS) methods, 2‐pool MT systems behave as a single‐pool with an apparent M0 and T1 that depends on the average RF power level (here parameterized via the RMS RF magnetic field (B1): B1rms),0 and T1 across measurements if B1rms is held fixed. Under this newly defined sampling regime, the parameters M0 and T1 derived from single‐pool relaxometry become explicitly dependent on B1rms, but may then be reliably reported along with the associated B1rms. This paper articulates and explores this framework and presents evidence to confirm that consistent and stable quantitative results can be achieved, paving the way for fast reliable relaxometry that may be suitable for both clinical and research use.Extended data acquisitions are required to obtain sufficiently diverse measurements to fit the plethora of parameters involved in full multi‐pool models of tissue and this has limited their adoption in larger scale studies and in the clinic. A much more common strategy has been to use single‐pool models, in which signals from tissue at each imaged location are treated as if they may be characterized by a single set of parameters 22.1≤W¯> that assumes an average saturation occurring throughout the entire imaging acquisition. A similar approach has been previously used to compare macromolecular saturation of different pulsed strategies based on a RF duty‐cycle.W¯> via B1rms as it is commonly reported across different vendors as an SAR‐specific metric. The current work proposes that, for SS sequences where a dynamic equilibrium is established and the longitudinal magnetization for each pool returns to the same value at intervals of TR, a relationship between the background pool‐induced saturation and the observed recovery of the free pool magnetization (such that it is driven by newly defined parameters M0CSMT and T1CSMT) can be established. To achieve this, we build on the Bloch‐McConnell equationThe ability to obtain whole‐brain 3D quantitative images with 1 mm isotropic resolution in a clinically feasible time (~10 min),Δω0 is the off‐resonance frequency shift of the static polarizing magnetic field, γ is the gyromagnetic ratio, and B1 is magnitude of the RF magnetic field. The superscripts f and m identify, respectively, the free and macromolecular pools of magnetization, mk and κf are first order exchange rates between the 2 pools . Here, B1rms represents the RMS amplitude of an arbitrary RF pulse whose energy is spread over a repetition period TR, and G is the absorption line shape of the macromolecular pool dependent on its spin‐spin relaxation rate, R2m, and parameterized by the frequency offset δ. R2m is large enough to ensure that RF pulses purely saturate the macromolecular pool (and hence R2m is omitted from Equations (1)‐(4) and Figure δ. In this mathematical description, we refer to relaxation rate R1, which is the reciprocal of the relaxation time T1, as this notation occurs more naturally in the equations and improves readability. As we assume W¯ decoupled from the irradiation field, algebraic manipulation of Equations (1A)‐(1C), summarized in Appendix B1rms‐dependent relaxation rateFor a pulsed experiment with short TR, we postulate that the recovery amount of macromolecular longitudinal recovery is limited and hence, the effect of the instantaneous mean saturation rate can be approximated as a continuous wave irradiation equivalentand equilibrium magnetization.B1rms→∞ the known solution for a fully saturated systemthat suffice to describe the time evolution of the 2‐pool system. In the limit where dMzmdt can be considered constant. Such is the case for short TR SS conditions where, for a repetition period, dMzmdt≈0. Therefore, the 2‐pool system can be characterized as a single pool with the definition of proton density and spin‐lattice interaction extended to M0CSMT and R1CSMT.This demonstrates that it is possible to describe a 2‐pool MT system as a single‐pool if 2.2B1rms. Equations (2A) and (2B) imply that single‐pool relaxometry methods can be sufficient to precisely perform qMRI on 2‐pool systems with MT, provided the RF saturation conditions are held constant across any necesssary variations in FA and TR Hence, we seek a pulse design strategy that allows free choice of free‐pool rotation angle, αf, and TR while keeping the total macromolecular pool saturation≤W¯> constant. This can be achieved because αf is proportional to the time integral of the excitation field B1 whereas γB1rms2=1TR∫0τRFγ2B12tdt is an integral of B12 evaluated over the pulse duration τRF. Our solution is a non‐selective multi‐band pulse ), the total saturation ≤W¯> is held constant as long as the energy of this pulse (∫0τRFγ2B12tdt) does not change. The free pool FA is set by changing the magnitude of the central band, with the outer bands adjusted to keep pulse energy equal to a reference rotation αref parameterized via B1refTypically, to enhance sensitivity of the relaxometry methods to the parameters of interest, a finely tuned combination of flip angles (FAs) and TR are selectedquations A and  and balanced steady state‐free precession (bSSFP) sequences with a range of excitation FA . A fixed TR of 7 ms was used for all sequences. The resulting data were fitted using both the well‐known driven equilibrium single pulse observation of T1/T2 (DESPOT1/2)0 and T1 from SPGR data and makes use of the estimated T1 to estimate a second M0 and T2 from a bSSFP signal model. The joint system relaxometry (JSR) approach achieves the same outcome in a single estimation step. These methods are expected to differ only in precision while maintaining accuracy,lsqnonlin routine. The objective function was defined as the sum of the squared differences between the single‐pool model and the simulated signal intensity. The stopping criteria were a tolerance of 10−15 on the cost function value or a maximum of 500 iterations. DESPOT1 fitting used the conventional approach of linearization1 was obtained in this case only. A C++ module integrated within MRtrix (https://www.mrtrix.org/) software is currently under development and will be made publicly available on https://github.com/mriphysics.For single‐pool relaxometry to be a useful approach, the estimated parameters should be independent of the choice of measurements made and the fitting procedure used. In the previous sections, we have presented the theory that enables this in 2‐pool MT systems if 3.1T1CSMT obtained from the CW approximation and ground‐truth T2. Both non‐CSMT and CSMT (reference FA of αref=65∘) conditions were studied, each with RF durations of τRF=0.614ms. For non‐CSMT, B1rms is calculated from the specified free‐pool FA αf, whereas under CSMT, B1rms was kept fixed throughout all measurements. For each independent instance, Gaussian distributed noise with zero mean and SD 0.002 M0f was added to both the real and imaginary components of the simulated signal. Tissue parameters were extracted from Gloor et al.Monte Carlo simulations of 2‐pool pulsed MT model systems were performed based on Equations B9) and (B11) of Appendix 9 and , and the proposed CSMT pulses, where RF power is held constant. Correct transmit field knowledge was obtained via the AFI approach0 and B1 inhomogeneities are mitigated. To further explore potential inconsistencies, different subsets of the measured FAs were selected, and parameters were estimated for each data subset based on the JSR approach.Phantom validation was obtained on a spherical container filled with hair conditioner  that is known to exhibit substantial MT effects.1 varies as a function of B1rms. To achieve this, we used the CSMT approach to sample SPGR data for different B1rms which vary between 0.2 μT and 2.0 μT in increments of 0.2 μT on a single volunteer. T1 maps were then obtained based on the DESPOT1 method.In vivo validation sought to corroborate our phantom experiment. Hence, the same FA measurements were performed on 3 different healthy volunteers who each gave written, informed consent according to local ethics requirements. Furthermore, we obtained data to validate the prediction that Twww.fsl.fmrib.ox.ac.uk) operating on the SPGR image with highest T1 contrast. The obtained masks were eroded based on a spherical element, radius 6 voxels, to create subject‐specific anatomic regions of interest (ROI) of ~2000 voxels, which were used for a summary comparison of parameter estimates for all FA subsets across all subjects.WM and gray matter (GM) masks were obtained for each subject's data using the FSL5.0 FAST tool . Both effects are removed under CSMT conditions , DESPOT2 (orange), and JSR (yellow) estimates were generated by model fitting to all the data Figure A‐D.2 estimations . Furthermore, for both T1 and T2, the results are more consistent across subsets as the observed RMS difference between the superset  and each subset is always lower under CSMT conditions as summarized in Table Figure 0 and T1 recovery parameters are expected to vary with B1rms. This is experimentally highlighted in Figure 1 maps obtained from measurements at different constant values of B1rms, demonstrating a clear change in this estimated parameter. The same effect was observed on M0 estimation.Furthermore, we seek to validate our theoretical description where both M50 and T1 that are conventionally regarded as properties of the free pool only. The proposed approach makes explicit not only the existence of a M0CSMT and T1CSMT dependent on the single parameter B1rms  would be inconsistent with one another, resulting in fitted parameters that are sensitive to incidental details of the measurement conditions. Depending on specific details of different manufacturers’ software, B1rms is expected to vary even for sequences with nominally equal parameters. This might explain the range of estimated values across difference studies even though nominally similar methods are used. The estimated parameters are only consistent when B1rms is held constant, which is possible via the proposed the CSMT framework. The observed T2 is shown to be both independent of the saturation conditions, if measurements with consistent M0CSMT and T1CSMT are obtained, and to agree with gold‐standard spin‐echo measurementsThe presented framework allows a novel insight into the common understanding of Ms Figure , but alss Figure  and 5, uWe note this methodology goes against the current trend of knowledge where a single, tissue‐specific, recovery time is sought that will be common between both SS and non‐SS gold‐standard inversion recovery methods for relaxometry.B1rms→0, the 2 measurable recovery rates are not expected to agree ). As an example, using the same tissue parameters for WM as the numerical validation described above, we expect R1obs=1.09s-1 whereas R1csmt=1.25s-1.A corollary of the CSMT framework is that SS methods sample a different interaction of the MT tissue model; even in the limit of R1obs is also not immune to MT saturation‐related inaccuracies.B1rms. For single‐slice or 3D imaging, this could be achieved experimentally by continuous application of off‐resonance saturation pulses during the sequence. From the derivation, it can be observed that full agreement  between non‐SS and SS recovery can only be obtained in the limit of full saturation of the macromolecular pool (B1rms→∞). Simulations based on literature WM 2‐pool MT parametersB1rms at maximum safety limits for deposited power.Although well‐established as a reproducible measure, T1CSMT dependency on B1rms in the in vivo experiment summarized in Figure T1CSMT values is shown for both WM‐ and GM‐specific ROIs. The presented data show that T1CSMT variations can be mitigated by operating in a low SAR regime  as there is limited T1CSMT variation between B1rms=0.2μT and B1rms=0.4μT. Hence the proposed CSMT framework is expected to have a significant impact of T2 estimation through SS sequences. However, shorter repetition times and higher FAs are typically required,B1rms and as a consequence, shorter T1CSMT values.We further highlight B1rms is easily controllable simply by adjusting TR, at the expense of acquisition time. However, such a naive approach would force sub‐optimal parameter choices in terms of overall estimation precision.B1rms. For slice‐selective applications, dedicated pulse design strategies need to be used where multiple FAs can be achieved at constant B1rms. As an example, one can easily envision a simultaneous multi‐slice acquisition where several slices are obtained simultaneously with different FAs to ensure CSMT conditions while maintaining acquisition time efficiency. In Figure B1rms impacts the in vivo measured T1CSMT. Although this appears to be an unfamiliar result at first, the range of obtainable relaxation times is in accordance with the panoply of relaxation times found throughout the literature, where relaxometry methods using only SPGR measures have reported WM T1 values between 801 ms and 1735 msB1rms must be reported, especially in measurements from multi‐center studies where absolute reported relaxation parameter values that characterize the measured object are sought. Perhaps even a standardized fixed power level should be agreed on by the community.For SS methods, The proposed framework is valid as long as our initial assumption of CW‐equivalent saturation holds. To explore the limits of such assumption, a numerical simulation was performed where SS obtained for a bSSFP sequence is compared for both pulsed saturation and CW equivalent (non‐pulsed). This is highlighted in Supporting Information Figure B1rms on the saturation achieved in the macromolecular pool. From Equations (5) and (6), it is expected there will be a residual spatial variation in the final estimated M0CSMT and T1CSMT maps because of this effect, although not in the determination of T2. This effect is minimized at low field strength (≤1.5 T) where spatial variations of transmit field are less—at high field strengths , alternative solutions are needed, and this is a focus for future research. From the measured spatial variation in RF amplitude of the experiment reported in Figure T1CSMT of 16% at 3 T.Transmit field corrections are necessaryquations  and (6),6B1rms in non‐selective SS sequences. However, this is just an optimized methodology for brain imaging, and slice‐selective approaches with CSMT conditions can be achieved by more conventional methods with separate off‐resonance pulses, provided B1rms is held constant for all measurements and is quoted as a key parameter in any reported results. In this work, we have operated at an arbitrary value of 2.06 µT, however, because B1rms can be simply determined by time integration of the RF pulses used in acquisition sequences, an agreed value can be achieved while still allowing latitude for differences in implementation details that may be system or manufacturer specific. A central consequence of the CSMT approach is that the measured proton density and longitudinal relaxation time both depend on B1rms as both are measures of the underlying multi‐compartment nature of the tissue concerned. This is a clear deviation from a frequent current interpretation of M0 as the magnetization of the visible pool when fully relaxed and T1 as a spin‐lattice relaxation time that excludes magnetization transfer. This change may take some getting used to, particularly as the absolute values of the quantities concerned can be quite different when there are substantial MT processes operating within tissues. The use of M0CSMT(B1rms), T1CSMT(B1rms), and T2 as quantitative measures to efficiently characterize tissue properties in health and disease remains to be proven in large scale studies, but the wealth of literature that explicitly accounts for MT supports their usage.This article presents a framework that enables consistent results to be obtained from single‐pool relaxometry in the presence of confounding magnetization transfer effects, which are ubiquitous in human tissues. This has the potential to reduce the currently observed inconsistency between relaxometry methods without requiring any more data than are currently acquired. The approach is compatible with a diverse range of existing high resolution in vivo relaxometry methods, and the data presented provides the first evidence that it can lead to stable and consistent results. Here, we made use of dedicated RF pulses that allow flexible balance between off‐ and on‐resonance energy to obtain at fixed This work received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/20072013/ERC grant agreement no. [319456] dHCP project) and MRC strategic grant [MR/K006355/1]. The research was supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King’s College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. This work was additionally supported by the Wellcome/EPSRC Centre for Medical Engineering at King’s College London [WT 203148/Z/16/Z].FIGURE S1 Comparison of measured signal (top), free pool longitudinal magnetization (middle) and macromolecular longitudinal magnetization (bottom) between pulsed and non‐pulsed bSSFP steady‐state for a fixed free pool flip angle of 68°.Click here for additional data file.


The detection and molecular analysis of circulating tumour cells (CTCs) potentially provides a significant insight to the characterisation of disease, stage of progression and therapeutic options for cancer patients. Following on from the protocol by Warkiani et al. 2016, which describes a method of enriching CTCs from cancer patient blood with inertial microfluidics, we describe a method to measure the CTC RNA expression in the enriched fraction using droplet digital PCR and compare transcript detection with and without RNA pre-amplification.•Inertial microfluidics combined with droplet digital PCR is advantageous as it allows for CTC enrichment and subsequent RNA analysis from patient blood. This allows for patient tumour analysis with increased sensitivity and precision compared to quantitative Real Time PCR and enables the direct quantification of nucleic acids without the need for tumour biopsy.•This method demonstrates an efficient approach providing important insights into the analysis of colorectal cancer patients’ CTCs using a specific gene subset or biomarkers, an approach that may be tailored to tumour type or expanded to larger panels. Specifications Tablein situ hybridisation (FISH), quantitative real time – polymerase chain reaction (qRT-PCR) and even single cell analysis as previously outlined [It has been well established that circulating tumour cells (CTCs) isolated from the blood of cancer patients provide the potential for non-invasive prognostic and molecular insights . Historioutlined ,11. As doutlined .Droplet digital PCR (ddPCR) is advantageous as it has been shown to have increased sensitivity and precision over traditional qRT-PCR, providing direct quantification of nucleic acids from a wider range of samples . ImportaFollowing on from the published protocol by Warkiani et al. which describes a method of enriching CTCs from patient blood with inertial microfluidics , we desca)9 ml blood samples were collected  in a cell free DNA BCT tube , stored at room temperature and processed within 24 h.b)Red blood cells were lysed using a commercial lysis buffer  with 35 ml of lysis buffer used per 9 ml of blood. Samples were placed on a gentle shaker for 15 min at room temperature before centrifugation for 10 min at 500 g with a slow brake.c)Supernatant was removed from the cell pellet and the pellet resuspended in PBS  -block-poly(propylene glycol)-block-poly)ethylene glycol). The pellet was resuspended in 3× volume of PBS than initial blood volume (i.e. 9 ml blood in 27 ml PBS).d)Microfluidic device enrichment was performed as described in Warkiani et al. . Brieflye)The enriched fraction was collected and pelleted with centrifugation for 10 min at 500 g.f)The supernatant was removed, the cell pellet resuspended in in 500 μl of RNA later and stored at −80 °C until analysis.CTCs were isolated using a trapazoidal spiral inertial microfluidic device as previously described . The traNote: Streck cell free DNA BCT tubes were used for this purpose as they contain a light preservative which allows for storage and shipping of samples without significant cellular degradation .a)g, and the RNAlater removed.The enriched sample (in RNA later) was centrifuged for 10 min at 3,500 b)100 μl of extraction buffer was added to the pellet as per manufacturer’s instructions  and was mixed well.c)The sample and extraction buffer was incubated for 30 min at 42 °C.d)g.The Picopure column was activated by adding 250 μl of conditioning buffer for 5 min at room temperature. The column was then centrifuged at for 1 min 16,200 e)g.100 μl of ethanol (from the Picopure kit) was added to the 100 μl of extraction buffer (RNA mixture), the sample was mixed well, added to column and centrifuged for 2 min at 100 f)g.100 μl of washing buffer #1 from Picopure kit was added to column and centrifuged for 1 min at 8000 g)g. Next, 100 μl of washing buffer #2 was again added to column and centrifuged for 2 min at 16,000 g and then 1 min at 16,000 g.100 μl of washing buffer #2 from Picopure kit was added to column and centrifuged for 1 min at 8000 h)g.11.5 μl of elution buffer was then added to column, incubated for 1 min and centrifuged for 1 min at 16,000 i)1.5 μl of the 10x ezDNAse buffer, 1 μl of nuclease free water and 1.5 μl of the ezDNAse were added to 11 μl of RNA as per the manufacturer’s instructions . Sample was incubated for 2 min at 37 °C and then placed at 4 °C.j)RNA quantity was measured using the Qubit RNA HS Assay Kit as per the manufacturer’s instructions . New standards were prepared before each set of readings (10 μl of standard and 190 μl of buffer). 3 μl of ezDNAse treated RNA sample was added to 1 μl of dye and 196 μl of buffer and the concentration was read using a Qubit 2.0 fluorometer (Thermofisher).Note: The presence of genomic contamination was monitored when analysing extracted RNA samples. For our set-up it was found with the Picropure RNA isolation kit alone that occasionally there was significant genomic contamination. Therefore, various methods  for additional DNAse treatment were investigated. DNAse treatment time was also investigated and determined that 2 min was adequate to prevent genomic contamination. ezDNAse (Thermofisher) was selected as the best method to eliminate the issue of genomic contamination.a)As per manufacturer’s instructions, per sample 4 μl of 5x iScript advanced reaction mix, 1 μl of iScript reverse transcriptase  and 4 μl of nuclease free water was added to 11 μl of ezDNAse treated RNA.b)Sample was placed in thermocycler and was run as per the following protocol: 20 min 46 °C, 1 min 95 °C and held at 4 °C.Due to the low amount of CTCs in enriched samples, we investigated the use of pre-amplification to increase the detectable amount of target RNAs. Pre-amplification was performed with Taqman pre-amplification master-mix as per manufacturer’s instructions (Thermofisher Scientific). For each blood sample pre-amplification was performed on 10 μl of cDNA. In this instance, we amplified using probes from the selected panel of CRC markers: KRT-19, CEACAM5, AGR2, FDZ7 and LGR5.2O 1:5 for running with the ddPCR.Briefly for each sample, 25 μl of 20× Taqman preamp master mix  was added to 12.5 μl of probe mix , AGR2, FDZ7 in 95 μl Tris-EDTA (TE) buffer ) and then 2 μl of nuclease free water and 10 μl of cDNA was added. The sample was then placed in the thermocycler and run as per the following protocol (10 min 95 °C then 15 s 95°c and 4 min 60 °C repeated 9× and held at 4 °C). Pre-amplified solution was again diluted with dHTo investigate pre-amplification utility we firstly used 10 or 50 of the ATCC® CCL-229™ LoVo cells  either in the presence or absence of WBCs to simulate normal enriched conditions. To quantitate efficiency, we used the Qubit 2.0 fluorometer for measurement of high sensitivity (HS) RNA (Q32852) and single stranded DNA . However, the ssDNA measurement is only relevant if the sample does not have background WBC contamination (as the pre-amplification will only amplify targets spiked into the mix). Therefore, we determined the best way to measure efficiency was to use ddPCR. We directly compared the same sample before and after amplification and it was found that 20–100× more positive droplets were obtained after amplification . SubsequFor each probe, the optimum annealing/extension temperature was investigated with a temperature gradient. It was found that 1 min for 60 °C was optimum for all probes in the expected temperature range for the assay. The optimum concentration for each probe was also investigated and it was found that 1.1 μl of each probe per reaction gave maximal results.1For each well, 1.1 μl of probe , 11 μl of ddPCR supermix , 4 μl nuclease free water, 4.8 μl cDNA for a final volume of 22 μl was added and mixed well. The plate was sealed with Biorad Aluminium plate seal (#1814040) and the plate was centrifuged for 30 s at room temperature.2The plate was placed in the QX200™ AutoDG™ droplet generation robot (Bio Rad) and set up as per manufacturer’s instructions.3After droplet generation, the plate was placed into thermocycler (Eppendorf deep well thermocycler) as per instructions for ddPCR Probe assay . i.e. enzyme activation 95 °C for 10 min, denaturation for 15 s 94 °C, annealing/extension for 1 min 60 °C (denaturation/extension repeated 39 cycles), enzyme deactivation 98 °C and hold at 4 °C with a ramp rate of 2 °C/sec.4The plate was then placed in QX200 Droplet digital PCR system and run as an absolute quantification experiment as per manufacturer’s instructions.5Analysis was then performed in QuantaLife (Bio Rad).Initially, probes were run individually with cDNA from either 10 or 10,000 ATCC® HT-29  cells (or LoVo cells) and expression was quantified. Two probes were then used together per analysis sample enabling the simultaneous detection on the FAM and HEX channels, either KRT19 and CEACAM5 or LGR5 and AGR2/FDZ7. The copies/μl of the target was compared alone and in combination. It was determined that the use of two specific probes in combination did not affect copy numbers/μl and could be used for analysis.Note: FAM is considered a brighter fluorophore than HEX and therefore, probes were used to match expected expression levels i.e. high expressing KRT19 was run with HEX while CEACAM5 was run with FAM.Note: Multiplexing with more than 2 probes involves mixing different amounts of FAM and HEX probes in various proportions to enable detection. The use of 5 probe multiplexing was investigated, however, proved problematic and unreliable for this specific application (due to the extreme rarity of markers) and it was ultimately decided to limit to 2 probes per reaction.Note: It is understood that in ddPCR false positive droplets will occur in no-template control runs and for negative samples . It is therefore, important to determine false positive rates for each probe and no template controls are continually run to determine the presence of reagent contamination and other errors. False positive rates assume all sources, droplets, detection, assays and contamination. False positive rate is calculated as number of events per well and used to calculate the limit of detection. False positive rate is different for each probe and must be calculated independently. We initially investigated FDZ7 as a marker of CTCs, however, it was found that the expression of this marker was too high in WBCs to detect CTC expression, as has been previously described . TherefoNote: We observed that pre-amplification significantly increases the number of false positive events (except for CEACAM5 which remained low) and therefore, while there is increase in the number of positive droplets, there is also a higher threshold over which a well can be considered truly positive . TherefoBased on the false positive rate, the number of positive droplets required per well to be a considered a true positive . For CEACAM5 which has a false positive rate of 0.6%, the threshold to assess a positive well was 3 positive droplets for 95% certainty that the result is a true positive. For KRT19 which has a 7.4% false positive rate, the threshold to assess a positive well is 22 positive droplets for 95% certainty that the result is a true positive. For LGR5 which has a 7.4% false positive rate the threshold to assess a positive well is 22 positive droplets for 95% certainty that the result is a true positive. For AGR2 which has a 32.1% false positive rate, the threshold to assess a positive well is 96 droplets for 95% certainty. This number of droplets is unrealistic and it is likely that these samples do not represent a true negative, therefore, similar to FDZ7, AGR2 is not a feasible marker to aid in the detection of CRC CTCs in this instance, although its utility has however, previously been demonstrated as a marker for prostate cancer CTCs .Using a dilution series, as few as 12 LoVo cells could be detected in 1 well using the CEACAM5 probe . As low Experiments spiking both HT-29 and LoVo cells into equivalent numbers of WBC background that is typically obtained following inertial microfluidic enrichment were first performed. It was found that the detection sensitivity was reduced with the presence of WBCs. Moreover, the presence of WBCs considerably increased the number of false positives (hence increasing the number of positive droplets needed to be considered a true positive) and decreased the limit of detection. Low numbers of LoVo cells (20 cells) could be detected within a background WBC population from 9 ml of enriched blood.Blood samples from CRC patients were processed as per the described protocol and expression of CTC markers was measured (Supplementary Information Table 1). The presence of CTC specific markers (hence CTCs) was confirmed in some of the patient samples in 1 or multiple samples from the same patient. This study has shown that mRNA expression of genes specific to epithelial or tumor cells can be detected in colorectal cancer patient samples.This article has described a method to assess mRNA expression of genes specific to epithelial or tumour cells with ddPCR after inertial microfluidic enrichment of CTCs from patient blood samples. It is shown that this method can be used to detect colorectal cancer patient CTCs. The detection of potential CTCs with ddPCR is made possible through the initial size based enrichment with the inertial microfluidic device as originally described by Warkiani et al. .


The exclusive formation of acetaldehyde is directly identified spectroscopically and discussed in the context of the underlying reaction mechanism. In line with computational predictions it is the terminal P=O and not the V=O unit that provides the oxygen atom in the barrier‐free thermal C2H4→CH3CHO conversion. Interestingly, in the course of the reaction, the emerging CH3CHO product undergoes a rather complex intramolecular migration, coordinating eventually to the vanadium center prior to its liberation. Moreover, the spectroscopic structural characterization of neutral C2H4O deserves special mentioning as in most, if not all, ion/molecule reactions, the neutral product is usually only indirectly identified.The gas‐phase reaction of the heteronuclear oxide cluster [VPO Caught in the act: The gas‐phase reaction of the heteronuclear oxide cluster [VPO4].+ with C2H4 was studied by infrared photodissociation spectroscopy, which directly identified the formation of acetaldehyde and revealed the terminal P=O moiety as the active site of [VPO4].+ in the oxygen atom transfer (OAT) reaction with ethylene, in line with theoretical predictions. DFT calculations have provided insight into the mechanism of this OAT catalytic cycle and predicted the terminal Al−O and not the expected V−O unit as the active site of the catalyst.4].+ cluster oxide as well as its active site was only obtained much later by cryogenic ion‐trap vibrational spectroscopy of messenger‐tagged cluster ions.The identification of the active site(s) of single‐site catalysts—the so‐called “aristocratic atoms”N2O x=3,  may servN2O x=3,  may serv4].+/C2H4 couple. This heteronuclear oxo cluster has served as a model system of industrially important VPO catalysts4].+ brings about the selective conversion of C2H4 to form C2H4O.4].+ (1). Other isomers considered were not only predicted to lie >100 kJ mol−1 higher in energy than 1, also their calculated IR spectra did not match the experimentally recorded one.2H4→C2H4O conversion. The alternative OAT process involving the terminal V=O group of 1 was calculated to require too much energy to play a role at ambient temperature.Herein, we describe the spectroscopic characterization of the reaction of the [VPO3].+ ion and in particular the neutral OAT C2H4O .1 is produced as previously described,2H4 in He (pHe≈0.022 mbar) under multiple collision conditions in a linear radiofrequency ion trap. The reaction products obtained after 100 ms are shown in Figure 4,(C2H4)n].+ adducts with n=0–3. Formation of [VPO3].+ and its adducts with C2H4 is roughly 100 times less efficient. The present results are in qualitative agreement with the previous results obtained under single‐collision conditions, which also found that adduct formation is favored over O‐atom loss; however, adduct formation was less efficient, and obviously no adducts with n>1 were observed under single‐collision conditions.1 and C2H4 in Figure To this end, we revisited the OAT reaction of 4].+/C2H4 couple, the new DFT calculations  and the vanadium sites of the cluster (1→6), with the latter path being energetically clearly favored.Coordination of the incoming C3CHO ligand (containing the oxygen atom of the P=O unit) is coordinated to the vanadium center (1→→→5). The isomeric cluster 9, generated from 6, is approximately 115 kJ mol−1 less stable than 5. However, as the transition states leading to both 5 and 9 are located below the entrance channel 1+C2H4, both routes are energetically accessible. Interestingly, 5 and 9 are connected via transition state TS9/4 and TS4/5.In the course of the multistep OAT reaction, the global minimum corresponds to a complex in which the newly formed CH3CHO to produce [P(O2)VO].+ can take place from either 4, 5, or 9.Evaporation of CH2, do not bind efficiently to cations at the present ion‐trap temperature of 150 K, we exploit the fact that larger .+ adducts are formed, in which the additional C2H4 moieties are more weakly bound and function as messengers upon photoabsorption. This is demonstrated in the mass spectra shown in the bottom panels of Figure −1), and the difference spectrum  from this on‐resonance spectrum. Using sufficiently attenuated laser pulse energies, photodissociation is only observed for the n>1 adducts, that is, the corresponding peaks are depleted in the on‐resonance spectrum n−1 type complexes are present, containing a single, chemically transformed ethylene species, while the others remain physisorbed.In order to identify the structure of the reaction product(s), we turn to infrared photodissociation (IRPD) spectroscopy combined with messenger tagging to ensure probing in the linear absorption regime; this simplifies the interpretation of the IRPD spectra significantly.4,C2H4].+⋅(C2H4) is shown in Figure 4].+⋅He2 , which lies in the carbonyl stretching region.The IRPD spectrum of .+⋅ (C2H4), we compare it to the B3LYPD2/def2‐tzvpp harmonic spectra of possible structural candidates in Figure 5, which is predicted to lie 283 kJ mol−1 below the entrance channel and represents the final reaction product containing acetaldehyde bound to [P(O)2VO].+. The exclusive formation of 5 rather than 4 or 9 , but did not observe any substantial photodissociation of untagged .+. Note that the predicted dissociation energy of 5 is 283 kJ mol−1, which would amount to the absorption of roughly 20 photons at 1000 cm−1, which is unlikely under the present experimental conditions. This shows that for bare [VPO4].+, chemisorption of C2H4 is highly favored over physisorption, and therefore the influence of the second C2H4 molecule is probably negligible under the present conditions.Using C4].+ to C2H4 is found in the IRPD spectrum of another product ion, namely that of (C2H4)n‐tagged [VPO3].+. Although the yield of [VPO3].+⋅(C2H4)1–2 is small compared to that of .+⋅(C2H4) 2. The good agreement between the experimental and computational results  confirms that the P−O2−V=O, and not the O=P−O2−V structure is generated upon desorption of CH3CHO from .+.Additional and independent spectroscopic support that the P‐center represents the active site for the OAT from [VPO4].+ for the reaction of [VPO4].+ with C2H4. Moreover, while in most ion/molecule reactions studied, characterization of the neutral product is based on circumstantial evidence,3CHO.In summary, the present study has experimentally confirmed the previously predicted mechanism, which postulated that the P‐atom represents the active site of the heteronuclear cluster [VPOThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The aggregation of α-synuclein (αS), a protein abundant at presynaptic terminals, is associated with a range of highly debilitating neurodegenerative conditions, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Emerging evidence indicates that the interaction of αS with lipid membranes defines both its physiological function and pathological effects. The characterisation of the modes of membrane binding by αS is therefore crucial to clarify the balance between normal and aberrant behaviour of this protein. Here we used solid-state nuclear magnetic resonance (ssNMR) spectroscopy to probe the nature of the N-terminally acetylated form of αS (NTAc-αS) bound to synaptic-like lipid vesicles. This post-translational modification is prevalent for the physiological form of αS and modulates the binding to lipid bilayers. By probing the structure, dynamics and membrane topology of NTAc-αS, we found that N-terminal acetylation does not alter significantly the conformational and topological properties of the membrane-bound state of αS, despite increasing its propensity for binding. Taken together, our data and previous characterisations of the cytosolic state of NTAc-αS clarify that the role of the N-terminal acetylation is to regulate the binding affinity of αS for synaptic vesicles without altering the structural properties of the bound state. The aggregation of αS is strongly linked with Parkinson’s disease (PD)6, as its aggregates are the major constituents of Lewy bodies in PD patients9. Point mutations, as well as duplications and triplications, in the αS gene are also associated with familial forms of PD15, and fibrillar aggregates of the non-amyloid-β component (NAC) region of the protein (residues 61–95) are associated with Alzheimer’s disease16.α-Synuclein (αS) is a 14 kDa monomeric disordered protein preferentially found at pre-synaptic termini17, including the maintenance of pools of SVs at the synaptic termini22, the promotion of interactions between SVs26 and the assistance of SNARE formation during neurotransmitter release30. αS has also been shown to bind mitochondrial membranes, where it may have role in the mitigation of oxidative stress of mitochondria33. A common characteristic of the majority of these putative functions is that they all require the binding of αS to lipid membranes34. Indeed, the partition between membrane-bound and cytosolic forms of αS is tightly regulated in vivo35 and appears to be crucial for the mechanism of αS aggregation41 as well as the toxicity of its fibrillar oligomers42. Understanding the biological regulation of this interaction is therefore a major priority to clarify the balance between functional and dysfunctional aspects in this presynaptic protein.The function of αS is currently still debated. Several lines of evidence have indicated a possible role for this protein in the regulation of the trafficking of synaptic vesicles (SVs)45 to a conformation that is enriched in α-helical structure. This ordering process is promoted by 7 imperfect sequence repeats of 11 residues located in the first 90 residues of the protein sequence and encoding for amphipathic α-helical motifs45. The modular organization of the repeats provides αS with the ability to bind a variety of assemblies ranging from small detergent micelles to lipid vesicles and membranes47 using a multiplicity of distinct binding modes43.Upon membrane binding αS undergoes a transition from an intrinsically disordered monomeric protein50. Moreover, the N-terminal acetylation of αS52, which is found in both healthy individuals and PD patients, has been shown to induce stronger membrane affinity, as well as an increased propensity to adopt transient α-helical structures in the N-terminal region54, a property that is enhanced upon copper interaction56.Membrane binding by αS is modulated by some post-translational modifications (PTMs), including the phosphorylation of different residues in the sequence57, a full understanding of the structural and dynamical properties of the membrane-bound state of NTAc-αS is currently missing.As the N-terminally acetylated αS (NTAc-αS) is considered to be the physiologically relevant form, and because of the central role of membrane binding for both functional and pathological contexts, it is important to investigate how this PTM alters the structure and dynamics of the membrane bound state of αS. Although previous studies have analysed the interaction between NTAc-αS and lipid membranes by probing the properties of its unbound state44. Using an approach based on paramagnetic relaxation enhancement (PRE) effects, we investigated the impact of the N-terminal acetylation on the topological properties of the protein with respect to the lipid membrane. Our experiments elucidated the nature of the membrane-bound state of NTAc-αS and clarified further the role of this PTM on the regulation of the membrane interaction of αS.To answer to this key question, we here employed solid-state NMR (ssNMR) to directly probe the conformational properties of NTAc-αS at the surface of small unilamellar vesicles (SUVs) that mimic synaptic vesicles for composition and size44. These regions include the N-terminal portion of the protein, which spans the initial 25 residues and acts as the primary membrane-binding motif that adopts the conformation of a stable amphipathic helix anchoring the protein onto the membrane surface. It was possible to assign the ssNMR resonances of the segment 6–25 of this region44 and employ these data to refine its structural ensemble when bound to synaptic-like vesicles58. In addition to the rigid N-terminal anchor, a central ‘sensor’ region (residues 26 to 98) resulted to have enhanced relaxation properties in the ssNMR spectra, suggesting that it exists in equilibrium between multiple conformations at the membrane surface  were found to remain largely unstructured and unbound to lipid bilayers, with some residues establishing transient contacts with the membrane surface44.Previous magic angle spinning (MAS) ssNMR studies of non-acetylated αS identified three different regions of the protein having distinctive structural and dynamical properties in the membrane-bound stateace Fig. 44. The c13C-15N labelled NTAc-αS samples were mixed with DOPE:DOPS:DOPC (5:3:2 w/w) SUVs at a protein:lipid ratio of 1:6544 and pelleted via ultracentrifugation into 3.2 mm rotors for MAS ssNMR. 13C-13C dipolar assisted rotational resonance (DARR)59 spectra of membrane-bound NTAc-αS, which were measured at −19 °C to enhance the signal to noise under conditions where this lipid mixture adopts a gel phase60, revealed a number of resonances belonging to residues from the N-terminal region in a rigid bound state at the surface of the SUVs  spectra62 to characterise the highly dynamic protein regions of NTAc-αS bound to DOPE:DOPS:DOPC SUVs  of lipids that incorporate a paramagnetic centre into their chemical structure. The spatial vicinity of protein regions to this centre enhances the transverse relaxation of their NMR signals, thereby providing a map of the contacts between the protein and the spin. Four different types of paramagnetic lipids were used to obtain a map of the contacts between the protein and spins placed at different positions of the membrane, i.e. from the hydrophilic head groups to the last carbon of the hydrophobic tail  provided evidence of contacts between atoms of the N-terminal region of NTAc-αS and the membrane surface. The spectra showed selective paramagnetic broadening of 13C-13C DARR resonances for both aliphatic-aliphatic and aliphatic-carbonyl cross-peaks, and suggested the topology of an amphipathic helix laying parallel on the membrane surface ail Fig. . PRE meaace Fig. . We thenace Fig. , with th58.These data indicate mild levels of insertion of the N-terminal region of NTAc-αS into the lipid bilayer, a result that is consistent with PRE experiments previously performed under the same experimental conditions by using non-acetylated αS17. It has now become evident that a fundamental element for the biological activity of αS is the interaction with lipid membranes66, which appears to be crucial in defining the balance between normal and aberrant forms of this protein. The membrane-binding affinity by αS is primarily influenced by the properties of the lipid bilayer, including curvature, charge, packing defects and surface hydrophobicity69, but other factors such as calcium binding70, phosphorylation50 and N-terminal acetylation52 have also been shown to modulate this interaction.Although close links between PD and the aggregation of αS are widely recognised, the physiological role of this presynaptic protein remains largely unclear56, and characterising its influence on the modes of membrane binding is crucial to understand biological behaviour of this protein. Previous solution NMR studies have probed this interaction by studying the properties of the unbound state of NTAc-αS in equilibrium with its membrane-bound form54. The resulting spectra showed increased relaxation properties for the resonances of the N-terminal region of the protein indicating stronger membrane-binding for this region when the protein is acetylated. This result is consistent with the present chemical exchange saturation transfer (CEST) experiments that enabled to characterise the equilibrium between bound and unbound states at a residue-specific level .αS was expressed and purified as previously described15N and/or 13C labeled αS variants were obtained by growing the bacteria in isotope-enriched M9 minimal media containing 1 g.L−1 of 15N ammonium chloride, 2 g.L−1 of 13C-glucose  and 1 g of protonated IsoGro 15N-13C . The growth was obtained at 37 °C under constant shaking at 250 rpm and supplemented with 100 μg.ml−1 ampicillin to an OD600 of 0.6. The expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 4 h, and the cells were harvested by centrifugation at 6200 g . The cell pellets were resuspended in lysis buffer  and lysed by sonication. The cell lysate was centrifuged at 22,000 g for 30 min to remove cell debris. In order to precipitate the heat-sensitive proteins, the supernatant was then heated for 20 min at 70 °C and centrifuged at 22,000 g. Subsequently streptomycin sulfate was added to the supernatant to a final concentration of 10 mg.ml−1 to stimulate DNA precipitation. The mixture was stirred for 15 min at 4 °C followed by centrifugation at 22,000 g. Then, ammonium sulfate was added to the supernatant to a concentration of 360 mg.ml−1 in order to precipitate the protein. The solution was stirred for 30 min at 4 °C and centrifuged again at 22,000 g. The resulting pellet was resuspended in 25 mM Tris-HCl, pH 7.7 and dialyzed against the same buffer in order to remove salts. The dialyzed solutions were then loaded onto an anion exchange column  and eluted with a 0–1 M NaCl step gradient, and then further purified by loading onto a size exclusion column . All the fractions containing the monomeric protein were pooled together and concentrated by using Vivaspin filter devices . The purity of the aliquots after each step was analyzed by SDS-PAGE and the protein concentration was determined from the absorbance at 275 nm using an extinction coefficient of 5600 M−1 cm−1. Mass spectrometry was used to assess that the level of N-terminal acetylation was complete.After transforming in BL21 (DE3)-gold cells , uniformly 44. Briefly, the lipid mixture was evaporated under a stream of nitrogen gas and then dried thoroughly under vacuum to yield a thin lipid film. The dried thin film was then re-hydrated by adding aqueous buffer  at a concentration of 15 mg.ml−1 (1.5%) and subjected to vortex mixing. In all NMR experiments described in this paper SUVs were obtained by using several cycles of freeze-thawing and sonication until the mixture became clear44. For ssNMR studies αS was added to the SUV mixtures up to a molar ratio of 1:65 protein:lipid. The mixtures were then pelleted at 303,747 g for 30 min at 4 °C  by using a TLA 100.3 rotor. Subsequently the SUV-αS samples were transferred into 3.2 mm Zirconia XC thin-walled MAS rotors for ssNMR experiments.Small unilamellar vesicles (SUVs) containing DOPE:DOPS:DOPC at a ratio of 5:3:2 (w/w) or POPG  were prepared from chloroform solutions of the lipids as described previouslyFree probe. Dipolar assisted rotational resonance (DARR) experiments59 were performed at a MAS rate of 10 kHz using a mixing time of 50 ms and a contact time of 1 ms. DARR spectra were acquired at −19 °C to favour signal to noise by inducing a gel phase of the lipid membranes. Previous studies showed that the chemical shifts of αS bound to DOPE:DOPS:DOPC SUVs at −19 °C and 4 °C are indistinguishable, ruling out conformational changes of the protein as a result of the different phases of the lipids44. Insensitive nuclei enhanced by polarization transfer (INEPT) spectra62 were measured at 4 °C using a MAS rate of 10 kHz. Pulse widths were 2.5 μs for 1H and 5.5 μs for 13C, and 1H TPPM decoupling was applied at ωRF/(2π) = 71.4–100 kHz44. 1H detected 1H-15N-HSQC of membrane bound NTAc-αS was measured as in Gopinath et al.63. Assignments of the resonances of NTAc-αS in DARR and INEPT spectra were derived from our previous study of αS bound to SUVs or unbound αS44.MAS ssNMR measurements were used to directly probe the resonances of NTAc-αS in the vesicle-bound state, which are inaccessible to solution-state NMR. MAS experiments were carried out on a 700 MHz Bruker Spectrometer with a 3.2 mm E75 probed the equilibrium between membrane unbound and membrane bound states of αS via direct detection of saturation in the resonances of the unbound state. In studying αS-SUV interactions, CEST shows higher sensitivity than measurements based on the signal attenuation in HSQC spectra, which enables measurements at low lipid:protein ratios to minimise αS or lipid aggregation44. Moreover, CEST signals are directly sensitive to the interaction between αS and the membrane surface and minimise the interference from additional factors that can contribute to the transverse relaxation rates of the protein resonances75. Solution state NMR experiments were carried out at 10 °C on a sample composed of αS (300 μM) incubated with DOPE:DOPS:DOPC SUVs at a concentration of 0.6 mg/ml and using a Bruker spectrometer operating at 1H frequencies of 700 MHz equipped with triple resonance HCN cryo-probe. CEST experiments were based on 1H-15N HSQC spectra by applying constant wave saturation in the 15N channel. Assignment of the solution NMR resonances was obtained from our previous studies44 and controlled with a series of 3D spectra by following a published protocol76. Since we aimed at probing the exchange between monomeric αS (having sharp resonances) and αS bound to SUVs (having significantly broader resonances), a series of large offsets was employed , resulting in CEST profiles of symmetrical shapes73. An additional spectrum, saturated at −100 kHz, was recorded as a reference. The CEST experiments were measured using a data matrix consisting of 2048  × 220  complex points.CEST measurements


On the other hand, this method allows metal oxides to disperse homogeneously. The cyclic voltammograms showed the stability of DSA, and the accelerated life test allowed establishing its useful life (18.18 years) at a current density of 10 mA cm−2.This paper describes a thermal method to obtain metal oxides on a titanium substrate surface. This adapted Pechini method is a versatile, easy to handle and scalable technique to obtain electrodes for industrial uses, such as Dimensionally Stable Anodes (DSA). This method has advantages over other thermal methods like dip coating or sputtering, as it needs a smaller amount of polymeric mixture than dip coating method to cover the same area and is less expensive than sputtering method. The thermal method described herein to prepare DSA type electrodes of RuO Specifications TableThe preparation of DSA type electrodes by this method consists of forming polyester from the reaction between ethylene glycol and citric acid, which allows dispersing the metals in a homogeneous way. Subsequently, by means of a thermal treatment, the organic matter is calcined, leaving the metal oxides supported on the Ti substrate. The method described in this work is an adaptation of the Pechini method   to remove chlorides from the titanium surface, and finally, 10-min ultrasonic cleaning (step 4).The Ti/RuO2-ZrO2 doped with Sb2O5) are: metal precursors and molar ratios employed, RuCl3 (0.0296), ZrO(NO3)2·H2O (0.0296), SbCl3 (0.0004), the polymeric precursor and molar ratios employed, ethylene glycol (16), citric acid (0.12), all reagents were analytical grade and the solutions were prepared with deionized water of 1 × 10−6 S cm−1 conductivity. If a different metal oxide is required, the precursor reagent of the metal of interest can be added. In this way we can adapt the method to obtain electrodes with a metallic oxide, a mixture of binary, ternary and even quaternary metal oxides.The reagents necessary to prepare the polymeric blend to ternary mixed metal electrode . Lastly, the blend was allowed to cool to room temperature. Then the titanium plates were coated with the polymer mixture, using a fine hair brush, such as camel hair (step 7), and heated in a Thermolyne Type 1500 muffle at 100 °C for 5 min (step 8); subsequently the plates were allowed to cool for 5 min (step 9). Steps 7, 8 and 9 (cycle A) were performed 8 times to later activate the electrodes in the muffle at 550 °C for 1 h (step 10) and finish a complete cycle (cycle B) with 8 layers of metal oxides on the titanium surface. The cycle B described above  was also performed 8 times to finally finish with 64 layers of metal oxides on the surface of the titanium.In the preparation of the polymer blend, ethylene glycol was heated to 60 °C, then citric acid was added until a homogenous mixture was obtained (with constant stirring) (step 5). The temperature was increased to 75 °C and RuCl2-ZrO2 doped with Sb2O5 electrode by X-ray diffraction and XPS was published by Rodriguez et al. [2 as dispersing agent and Sb2O5 as dopant in the oxide mixture. EDS surface images exhibit a uniform dispersion of the analyzed elements, The characterization of Ti/RuOz et al. , where t2-ZrO2 doped with Sb2O5 electrode was tested with cyclic voltammetry at 20 mV s−1 in a potential range of 0.5−1 V vs. SHE. The system used consisted of a three-electrode cell, where the anode was the ternary electrode, cathode was a graphite rod and the reference electrode was a saturated calomel electrode, and the electrolyte was a solution (50 mL) containing 4 mM K4Fe(CN)6 and 1 M NaNO3. −1) that are very similar, which means that the properties of the oxides do not change with the applied potential.The stability of Ti/RuO2-ZrO2 doped with Sb2O5) with 1 cm2 surface area was used as the working electrode and a graphite rod as the counter electrode, whereas the reference electrode was a saturated calomel electrode equipped with a Luggin capillary. 1 M NaCl was used as supporting electrolyte, and the cell temperature was controlled at about 25 °C. A power supply provided a constant anodic current density of 0.5 A cm−2. The solution was replaced each 24 h and the potential of the working electrode was periodically monitored. EA and jEA are service life and current density of electrolysis application, SLa and ja are the service life and current density under the accelerated life test conditions. The estimated useful life of the prepared electrode is 18.8 years, which is competitive for industries.Accelerated life test was performed in order to estimate the durability of the DSA electrode. A typical three-electrode cell was used; the DSA (Ti/RuO


We report on catalysis by a fuel‐induced transient state of a synthetic molecular machine. A [2]rotaxane molecular shuttle containing secondary ammonium/amine and thiourea stations is converted between catalytically inactive and active states by pulses of a chemical fuel (trichloroacetic acid), which is itself decomposed by the machine and/or the presence of additional base. The ON‐state of the rotaxane catalyzes the reduction of a nitrostyrene by transfer hydrogenation. By varying the amount of fuel added, the lifetime of the rotaxane ON‐state can be regulated and temporal control of catalysis achieved. The system can be pulsed with chemical fuel several times in succession, with each pulse activating catalysis for a time period determined by the amount of fuel added. Dissipative catalysis by synthetic molecular machines has implications for the future design of networks that feature communication and signaling between the components. An out‐of‐equilibrium state of a synthetic molecular machine is used to control catalysis. A rotaxane is transiently converted from a catalytically inactive state into an active form by CCl3CO2H. Deprotonation by the rotaxane promotes decarboxylation of the acid, thereby returning the system to its original state and stopping catalysis. This process allows temporal control of a coupled biomimetic reduction reaction. Living systems are complex dissipative cellular networks that are capable of advanced functions such as adaptability, responsiveness, and evolution through the consumption of energy.To date, the only example of an artificial system where catalytic activity is turned on by the presence of a chemical fuel is a vesicle system developed by Prins and co‐workers.1  ammonium and (weakly binding) amine groups.1H+][CF3CO2−] was prepared in six synthetic steps from commercially available materials through a threading‐and‐capping strategy (see the Supporting Information). Deprotonation of the rotaxane was achieved with polymer‐supported 2‐tert‐butylimino‐2‐diethylamino‐1,3‐dimethylperhydro‐1,3,2‐diazaphosphorine (BEMP) phosphazene base in CH2Cl2 to form neutral 1. The translocation of the macrocycle between the different sites in 1 and 1H+ was established by comparison of their 1H NMR spectra in CD2Cl2 . Similar chemical‐shift changes occur in [D8]toluene, which proved a more convenient solvent for subsequent experiments with the chemical fuel.Rotaxane H+toluene immediately protonated the amine group, with 1H NMR showing similar shifts to [1H+][CF3CO2−], thus confirming translocation of the macrocycle from the thiourea to the newly formed ammonium group . After 9 h, the amount of 1H+ present started to decline, gradually converting back1 over the course of 7 h.1H NMR spectrum of the reaction mixture was identical to that of the starting material apart from the additional presence of the CHCl3 waste product. To demonstrate the robustness of the dissipative cycling, a series of fuel pulses were made to the same sample . Each time (for at least seven fuel cycles), rotaxane 1 underwent switching, with no decomposition of 1 nor any fuel‐induced fatigue apparent by 1H NMR spectroscopy.Addition of CCl1H+ state could be varied by pulsing with different amounts of CCl3CO2H.1 in less than 72 h, whilst addition of 1.9 equiv extended the time required to 7 days. With 2.5 equiv of fuel, more than 2 weeks were needed for the rotaxane to return to its original state. The addition of triethylamine significantly accelerated the fuel decompositionThe lifetime of the 3 through Hantzsch ester based transfer hydrogenation3CO2H.tert‐butyl ester 4 as the Hantzsch ester hydrogen donor  to give reduced product 5 was monitored in the presence of a range of additives . With a starting concentration (c0) of 3 of 0.05 m, the background initial rate was 0.20 mm h−1 (ca. 0.05 mm h−1 at c0=0.025 m), giving 8 % conversion of 3 and 4 to 5 over 24 h (2 % with c0=0.025 m). The presence of Et3N or CCl3CO2H caused negligible rate enhancements.5 being formed in 50 % yield after 24 h .The reaction between 1 and 1H+ was then examined . A reaction rate of 0.044 mm h−1 between 3 and 4 (c0 [3]=0.025 m) was measured in the presence of 15 mol % OFF‐state catalyst 1, which is virtually the same as the background. Under similar conditions, the ON‐state, 1H+, induced a 6‐fold increase in reaction rate to 0.28 mm h−1.1 (15 mol %) and Et3N  were added to 3 and 4 in [D8]toluene. After 20 h, a pulse of CCl3CO2H (0.20 equiv) was added, switching 1 to the ON‐state (1H+). 1H NMR was used to confirm translocation of the macrocycle from the thiourea to the dibenzylammonium site. After the fuel had fully decarboxylated , 1H NMR again confirmed that the rotaxane catalyst had reverted back to the inactive state. The effect on the fuel‐induced catalysis of the reaction between 3 and 4 by the rotaxane is shown over one cycle under conditions that optimize the conversion to 5 in a single pulse  to study the effect of multiple fuel pulses. Stepwise increases in the amount of 5 formed were apparent over three successive fuel pulse cycles, each lasting 12–18 h .The dissipative catalysis is more pronounced at higher concentrations  should be addressed to the authors.SupplementaryClick here for additional data file.