The grafting of modifier on the surface of ZrO2 was confirmed by Fourier transform infrared spectroscopy and thermogravimetric analysis (TGA). Finally, the resulting modified ZrO2 (ZrO2–VB1), was used as a nano-filler and incorporated into the PVC matrix to improve its mechanical and thermal properties. These processes were carried out under ultrasonic irradiation conditions, which is an economical and eco-friendly method. The effect of ZrO2–VB1 on the properties and morphology of the PVC matrix was characterized by various techniques. Field emission scanning electron microscopy and transmission electron microscopy analyses showed a good dispersion of fillers into the PVC matrix with the average diameter of 37–40 nm. UV–Vis spectroscopy was used to study optical behavior of the obtained NC films. TGA analysis has conﬁrmed the presence of about 7 wt% VB1 on the surface of ZrO2. Also, the data indicated that the thermal and mechanical properties of the NC films were enhanced.In the present investigation, solution casting method was used for the preparation of nanocomposite (NC) films. At first, the surface of ZrO NCs obt2 NPs has a great deal of attention, because of their properties such as high thermal stability, high oxygen ion conductivity, high refractive index and band gap [Excellent chemical resistance, easy modification, nonﬂammability and low production cost of poly(vinyl chloride) (PVC)  make PVCband gap .2 NPs can be directly incorporated into the polymeric matrixes, but because of NPs aggregation which resulted by incompatibility of them within the organic polymers and their specific surface area, it is arduous to produce homogeneous dispersion into the polymer matrix [ZrOr matrix .2 NPs with suitable compatibilizers is introduced as an efficient strategy to overcome this problem [To get better dispersion stability of NPs into the polymer matrixes, surface modifications of ZrO problem . Various problem .1 (VB1) is a colorless, crystalline, bio-safe, low cost and organosulfur biological compound that is soluble in water and practically insoluble in less polar organic solvents. VB1 has several functional groups such as hydroxyl and amino groups that can act as ligand to hydroxyl groups on the surface of ZrO2 NPs [1 for chelate to ZrO2 NPs makes it a good candidate as modifier for surface modification of ZrO2 NPs [Vitamin BZrO2 NPs ,15. The 1) was chosen for the surface modification of ZrO2 NPs in order to achieve excellent dispersion and improve interface between ZrO2 NPs and PVC matrix. Finally, the modified ZrO2 (ZrO2–VB1) was integrated into the PVC matrix to improve its thermal, morphological, mechanical and optical properties. All the processes have been carried out by an ultrasonic technique. Ultrasonic irradiation has been widely used in medicine, chemistry and preparing NCs for control size distribution, activation of particles and decreasing the aggregation in the polymer matrix [In this work, first a bioactive coupling agent , UV–Vis spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM) and contact angle measurement (surface wettability properties). Finally, tensile tests were performed to characterize the NCs mechanical behavior.2.2.1.−1 and scanned at wavenumber range of 400–4000 cm−1. FT-IR spectra were used to characterize the chemical bonds of the prepared samples in KBr pills for powder materials and spectrograms of NC films were acquired directly. The XRD patterns were used to characterize the crystalline structure of the samples. The XRD patterns were acquired by using a Philips X’Pert MPD X-ray diffractometer (Germany). The scans were obtained using Cu Kα incident beam (λ = 1.5418 Å) at current of 100 mA and voltage of 40 kV with 2θ ranging from 10 to 80° and scanning rate of 0.05° min−1 for films and powder specimens. The UV–Vis spectra of the NC films were measured by UV–Vis–Near IR spectrophotometer JASCO, V-570, with solid samples of NC films in the spectral range between 200 and 800 nm. The dispersion morphology of modified NPs and NC films were studied by FE-SEM analysis  and TEM images by a Philips CM 120 microscope operated (Netherlands) at voltage of 100 kV. TGA data were done with a STA503 system of TA instrument (Germany) with 20 °C/min rate of heating under argon atmosphere. The temperature was varied from room temperature to 800 °C. In order to test films mechanically, tensile testing was carried out with the speed of 5 mm/min on a Hounsfield test equipment H25KS  with a load cell of 60 N at room temperature. The dimensions of the test samples were 35 mm × 9 mm. Contact angle measurement were done with a U-VISION MV500 digital camera microscope (China) .The reactions were performed at room temperature by a TOPSONIC ultrasonic liquid processor (Iran) with power of 400 W in wave frequency of 20 kHz. FT-IR spectra of the specimens were recorded on a Jasco-680 spectrophotometer (Japan) at a resolution of 4 cm2.2.2 powder was purchased from Neutrino Co. (Iran) with average particle sizes of < 50 nm. Vitamin B1 (Mw: 300.81 g/mol) was supplied from Alfa Aesar Chem Co. PVC  and THF (Mw: 72.11 g/mol) were obtained from LG Chem Co. (Korea origin) and JEONG Wang Co. (Korea).Nanosized ZrO2.3.2 NPs in the PVC matrix, the surface modification of ZrO2 NPs must be performed. For the modification of ZrO2 NPs with VB1: 0.01 g of VB1 was added into 8 mL of deionized (DI) water and was ultrasonicated for 15 min. Next, 0.1 g of ZrO2 NPs was dispersed in 8 mL of DI water by sonication for 15 min to obtain a suspension of ZrO2 NPs. Then, the VB1 solution was added to the ZrO2 suspension and was sonicated for 30 min under ultrasonic radiations to obtain stable suspension. After irradiation, the mixture was centrifuged, washed thoroughly with water, and dried at room temperature to give the product of ZrO2–VB1 NPs. The reaction sequence for the functionalization of ZrO2 NPs with VB1 is shown in Scheme For better homogeneous distribution of ZrO2.4.2–VB1 NC films were prepared via solution casting, using ultrasonic irradiation method. In this procedure, 0.1 g of PVC was first dissolved in 5 ml of THF and stirred using a magnetic stirrer at 40 °C for 1 h to achieve a homogenous solution. Then, ZrO2–VB1 NPs with different contents  were added to PVC solution and stirred for 12 h at room temperature. Finally, the obtained mixtures were ultrasonicated for 30 min and the resulting homogeneous mixtures were casted onto glass Petri dishes and dried at room temperature to prepare NC films. All of these stages were shown in Scheme The PVC/ZrO2–VB1 NPs are shown in Figure The photographs of the prepared NC films with different contents of ZrO3.3.1.2 NP is one of the most interested inorganic nano-filler that exhibits advantages such as UV filtering and thermal stability when inserted in a polymer matrix [2 NPs and high surface area cause zirconia aggregates [2 NPs is an efficient strategy to overcome this problem [1, it was used as a modifying agent. Due to the presence of numerous hydroxyl and amino groups in the VB1, it generates more functional groups and active sites on the ZrO2 NP surface. Finally, ZrO2–VB1 NPs are used for the synthesis of PVC/ZrO2–VB1 NC films. In fact, the driving force for the adsorption of ZrO2–VB1 onto the PVC surface is hydrogen bonding. Also, Van der Waals forces between ZrO2–VB1 NPs surface and PVC segments can be responsible for the overall adsorption process [ZrOr matrix . Thus, igregates . Surface problem . Because process . Ultraso process .3.2.2, ZrO2–VB1 NPs and VB1 as coupling agent are shown in Figure 2, the absorption bands at 3448 and 1630 cm−1 were corresponded to the stretching and bending vibrations of hydroxyl groups bands, which linked to the ZrO2 NPs [−1 was attributed to the Zr–O stretching vibration and a band at 1435 cm−1 was correspond to the bending vibration of Zr–OH [1 . Also, two bands around 1595 and 1615 cm−1 were assigned to the C=N and C=C stretching vibration bands in the pyrimidine ring for VB1 [2–VB1 NPs is shown in Figure −1 can be assigned to the NH group in VB1. It shows that NH vibration of VB1 near 1667 cm−1 is shifted to 1658 cm−1 in ZrO2–VB1. The reason of this observation is the existence of interaction between ZrO2 NPs and VB1. Furthermore, the broad band in region of 2800–3400 cm−1 can be related to different hydroxyl and amino groups of VB1 [2 NPs surface.FT-IR spectra of pure ZrOZrO2 NPs . The banof Zr–OH . In the  for VB1 . The FT-2–VB1 NC films. In the PVC spectrum, the absorption bands at 616 and 692 cm−1 were attributed to the stretching of C–Cl groups [−1 were related to the C–C, C–H, CH2–Cl and CH2 stretching vibrations, respectively. PVC/ZrO2–VB1 NC films , N–H (bending) and C=C groups of the modifiers that its intensity has been increased with increasing the modified filler content.Figure l groups  and 29713.3.2–VB1 NC films was fabricated by the interactions of –Cl groups in the PVC chains with –NH and –OH groups on the ZrO2–VB1 NPs. Figure 2–VB1, pure PVC and PVC/ZrO2–VB1 NC films with two different magniﬁcations. It is evident that NPs have homogenous shape. FE-SEM image of the PVC/ZrO2–VB1 NC films shows that the obtained macromolecules have nanostructures morphology. This phenomenon can be due to the using of ultrasonic technique [2–VB1 NPs.A high homogenous dispersion of the PVC/ZrOechnique . Accordi2–VB1, where the average size of NPs was around 37 nm, as calculated by Digimizer software and SPSS statistics. The images clearly demonstrated that the ZrO2–VB1 were dispersed in the water and did not show serious aggregation. Figure 2–VB1 NC 3 wt% at different magnifications. From the micrograph, it is observed that the modified NPs has uniform size distribution with an average size of 40 nm in diameter, almost spherical in shape and are well dispersed within the PVC matrix. This result is presumably due to the fact that the surface of ZrO2 with high surface energy was modiﬁed with VB1, which improved the compatibility of the ZrO2–VB1 with the matrix.These observations were also conﬁrmed by TEM images. Figure 3.4.2–VB1 are shown in Figure 2 is direct band gap insulator, photocatalyst, an active and typical photon absorber. The monoclinic crystalline structure of ZrO2 has two direct interband transitions at 5.93 and 5.17 eV. The pure ZrO2 NPs have a sharp and intense band at 212 nm with an absorption edge around 300 nm [λ = 210 and 280 nm, which can be assigned to the π–π* and n–π* transition, respectively. The significant increase in the absorbance below 256 nm is associated with the C–Cl bond [2–VB1 NPs, which can increase UV absorption of the NCs because of transfers and conjugate structure of modifier and UV-resistant property of ZrO2 NPs. Therefore, the prepared NCs can be used as a UV shielding material.UV–Vis spectra of pure PVC and the NC films with different amounts of ZrOd 300 nm . For the–Cl bond . As show3.5.2 and ZrO2–VB1 NPs. In decomposition processes, the weight loss below 120 °C was ascribed to physically adsorbed water [2–VB1 one step degradation can be seen at around 250 °C, which it may be ascribed to the separating of modifier from the surface of ZrO2 NPs. The amount of coated modifiers on the surface of ZrO2 NPs can be estimated by comparing the weight loss of pure ZrO2 NPs and modified ZrO2 NPs from the residue via TGA thermogram. Residual weight loss values at 800 °C for the pure ZrO2 NPs is 2 wt% due to the removal of physically adsorbed water and it is 9 wt% for modified ZrO2. So, the actual VB1 weight percentages supported on the NPs can be calculated and it is about 7 wt%.TGA analysis is an analytical technique used to determine a material’s thermal stability and its fraction of volatile components by monitoring the weight change that occurs as a sample is heated . Figure ed water . In ther1 on ZrO2 surface is approximately around 7 wt%. So, it can be concluded that the modifier was successfully grafted on the surface of NPs.As shown in graph, it can be readily predicted that the weight ratio of the grafted VB2–VB1 NC films of different wt% are shown in Figure TGA curves for the pure PVC and PVC/ZrO5) and 10% (T10) degradation occurred, char yield (residues of PVC/ZrO2–VB1 NC films at 800 °C) and the limiting oxygen index (LOI) based on Van Krevelen and Hoftyzer equation  [2 NPs have a higher decomposition temperature in contrast to the pure PVC. In fact, the presence of the ZrO2–VB1 could restrict the mobility and ﬂexibility of the PVC chains and lead to an increase in the decomposition temperature [2–VB1 except in the case of 7 wt% that may be due to the partial aggregation and reduce the effect of NPs on the polymer. It is reported that materials with LOI values more than 21 can show self-extinguishing behavior and considerate as flame retardants, so these NCs can be classified as the self-extinguishing materials [The temperature at which 5% , caused superior load transfer from the matrix to the reinforcement. In addition, compared to the pure PVC, the elongations at Fmax for NCs were increased with incorporation of ZrO2–VB1. The possible reason for this observation may be due to decreasing the intermolecular interactions density in the polymer chains and increasing the free volume between the polymer chains after incorporation of NPs which increase the flexibility of NCs [Figure 4.2 NPs was modified with a biosafe molecule (VB1). FT-IR and TGA analyses showed that modifier was grafted onto the surface of NPs. TEM and FE-SEM confirmed that NPs dispersed well after the surface modification. Then, the PVC/ZrO2–VB1 NC films were prepared by adding different amounts of ZrO2–VB1 NPs into the PVC matrix through solution casting method under ultrasonic irradiation as a green and an easy tool. The thermal, mechanical, morphology, wettability and optical properties of the PVC/ZrO2–VB1 NC films were determined. The morphology of the obtained NC films is not different from pure PVC and showed homogeneous dispersion of ZrO2–VB1 in the PVC matrix. TEM micrographs of the PVC/ZrO2–VB1 NC films revealed that maximum frequency of particle sizes was in the range of 36–40 nm. The results of thermal properties indicated that thermal stability of the PVC films is enhanced after incorporation of the ZrO2–VB1 NPs. PVC water absorption has increased by incorporating ZrO2–VB1. UV–Vis diagrams showed that NC films have more optical absorption than pure PVC. According to the XRD results, the modification process had no effect on ZrO2 crystalline structure. Also, mechanical tests indicated that the NC films were more flexible than the pure polymer. Therefore, the surface modification of ZrO2 NPs and its loading into the PVC matrix improved the polymer properties.In this study, the surface of ZrONo potential conflict of interest was reported by the authors.This research was financially supported by the Research Affairs Division Isfahan University of Technology (IUT), Isfahan, Iran.


We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC 4.1.1.1), which catalyzes the decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide.Dissolution dynamic nuclear polarization can increase the sensitivity of the Zymomonas mobilis (zmPDC) and a mutant that lacked enzyme activity were expressed using an inducible promoter in human embryonic kidney (HEK293T) cells. Enzyme activity was measured in the cells and in xenografts derived from the cells using 13C MRS measurements of the conversion of hyperpolarized [1‐13C] pyruvate to H13CO3–.Pyruvate decarboxylase from zmPDC expression in the cells and in the xenografts derived from them resulted in an approximately two‐fold increase in the H13CO3–/[1‐13C] pyruvate signal ratio following intravenous injection of hyperpolarized [1‐13C] pyruvate.Induction of zmPDC as an in vivo reporter gene for use with hyperpolarized 13C MRS. Magn Reson Med 76:391–401, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.We have demonstrated the feasibility of using  HEK293T cells were grown at 37°C in Dulbecco's Modified Eagle's Medium (DMEM)  with 10% fetal bovine serum , 4 mM glutamine (GIBCO Invitrogen) and 5% CO2. Plasmid DNA transfections were performed with Lipofectamine (Life Technologies Ltd) with a 10:1 molar ratio mixture of the tet‐on PDC plasmid and pPUR vector . Stably transfected clones were selected in media containing 2 μg/mL puromycin, picked, and expanded (see Supporting Information Section S2).A DNA sequence encoding a monomer of pyruvate decarboxylase from His Fig. . A variaTwenty‐five thousand cells were seeded per well in 48‐well plates , with six replicates per experimental condition. Eight hours after seeding, an appropriate volume of a 1 mg/mL solution of doxycycline in sterile water, or water alone, was added to the media. Confluence of the monolayer was measured using an IncuCyte instrument , which was then used to calculate the cell doubling time using Prism 6.0  .The growth medium was refreshed 24 h after cell seeding in T25 tissue culture flasks. For dose–response experiments, doxycycline was added to the media at the indicated concentrations, and the cells were harvested 48 h later. For time course experiments, the medium was supplemented with 2 µg/mL of doxycycline at 24, 48, or 96 h before cell harvesting.Supporting Information Section S3).Cells were lysed in passive lysis 5× buffer , diluted in water, and contained complete ethylenediaminetetraacetic acid–free protease inhibitor cocktail  as recommended by the manufacturer. Protein concentration was measured using a Bradford protein assay and a Sunrise 96‐well plate reader . The protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted onto a polyvinylidene difluoride membrane, and probed for expression of the V5 epitope and α‐actin . pyruvate in H2O was added to the cell suspension and mixed. After the addition of 100 μL of mineral oil, the sample was returned to the spectrometer and, after thermal equilibration, a series of 16 1H spectra were collected using a pulse and acquire sequence with 90° flip angle pulses . The cell pellets were lysed in 0.45 mL of ice‐cold lysis buffer, essentially as described above, except that the lysis buffer was diluted in 200 mM citrate buffer (pH 6.0). A 480‐μL sample of the lysate was then mixed with 60‐μL of a solution of 10 mM thiamine pyrophosphate and 10 mM MgCl2 in 2H20 and used for 1H MRS measurements.Sodium pyruvate was deuterated as described previously All animal experiments were performed in compliance with a project license issued under the Animals (Scientific Procedures) Act of 1986 and were designed with reference to the UK Coordinating Committee on Cancer Research guidelines for the welfare of animals in experimental neoplasia 7 cells stably transfected with the tet‐on PDC‐GFP‐V5/His plasmid expressing the wild‐type or mutant enzymes. Xenograft diameters were measured using calipers, and the length (L) and width (W) were used to calculate their volumes (V) according to the formula V = ½ (LW2) Each animal was injected subcutaneously in the left flank with 0.1 mL of a cell suspension containing 2.5 × 103 within 21–25 days from implantation. To induce PDC expression, a solution of 10 mg mL−1 doxycycline with 5% w/v sucrose was given to the mice in their drinking water ad libitum for 96 h prior to imaging experiments.The xenografts typically reached a volume of approximately 0.5–0.7 cm13C/1 H volume coil  in a 7T horizontal bore magnet (Varian). Xenografts were localized in transverse 1H images acquired using a spin‐echo pulse sequence . Immediately after dissolution, 10 µL/g body weight of the hyperpolarized pyruvate solution  was injected via the tail vein over a period of 2 s. Spectroscopic acquisitions were started 30 s from the injection. Single transient spectra  from an 8‐mm slice through the tumor were acquired. The center frequencies for the spectra were selected to be 175 ppm and 200 ppm for experiments with [1‐13C] pyruvate and [2‐13C] pyruvate, respectively. For experiments with [2‐13C] pyruvate, the protons were decoupled using a WALTZ decoupling sequence during acquisition (B1 ∼1.65 kHz), centered at 9.7 ppm.Animals were anesthetized and monitored as described previously g and 4°C in an Eppendorf 5415D bench top centrifuge  to remove debris.Freeze‐clamped xenograft tissues were homogenized using a manual Potter S homogenizer  in 4 volumes (milliliters per gram wet weight of tissue) of ice‐cold lysis buffer. Homogenates were centrifuged at 16,200 Supporting Information Section S5).Immunohistochemical protocols and image analysis were performed using instruments, reagents, and software from Leica  unless stated otherwise. Xenografts were excised and fixed in 4% neutral buffered formalin solution for 24 h and then transferred to 70% ethanol. After paraffin embedding, tissue sections were taken from the center of the xenograft and stained for cleaved caspase 3 (CC3), V5 epitope, and GFP expression. Hematoxylin and eosin (H&E) staining was performed to assess tissue viability  was identified manually and compared with the total xenograft area, and the result was converted to percentage cell death. The Aperio ImageScope Positive PixelCount v9 macro, tuned to identify negatively and positively 3,3’‐diaminobenzidine–stained tissue, was used for quantification of CC3 staining, which is reported as the number of positive pixels as a percentage of the total number of pixels.t test at the 95% confidence level.The results are presented as the mean ± standard deviation unless stated otherwise. Statistical significance was assessed using Microsoft Excel with a two‐tailed 13C T1s measured in vitro were 42.2 s for [1‐13C] pyruvate , 29.5 s for [2‐13C] pyruvate , 11.5 s for [1‐13C] acetaldehyde , and 40.7 s for bicarbonate  at 11.7T  than those measured at 9.4 T and 7 T, which were the magnetic field strengths used for the cell and xenograft experiments, respectively.zmPDC‐GFP‐V5/His transgene was detectable on western blots by 24 h after addition of 2 μg/mL of doxycycline to the growth medium and showed further increases at 48 and 96 h. Expression was dose dependent, showing an increase between 0.5 and 2.0 μg/mL doxycycline and was undetectable in the absence of the drug , demonstrating that the activity of the enzyme in the cells was limited by the rate of pyruvate transport. PDC activity was not detectable in cells that did not express zmPDC‐GFP‐V5/His protein or that expressed zmPDC H113Q‐GFP‐V5/His protein at 48 h after addition of 2 μg/mL doxycycline to the growth medium  pyruvate . Addition of hyperpolarized [1‐13C] pyruvate to cells resulted in a time‐dependent increase in the bicarbonate signal at 162.8 ppm, which could be followed using dynamic 13C MRS measurements  and mutant enzymes (zmPDC/H113Q‐GFP‐V5/His) following doxycycline administration; however, this decrease was not statistically significant , nor did the levels of apoptosis, as assessed by the levels of cleaved caspase 3 staining    or the total observable 13C signal . The total observable 13C signal included contributions from pyruvate, pyruvate hydrate, alanine, lactate, and bicarbonate. There was no measurable increase in the bicarbonate signal in xenografts expressing the inactive variant of zmPDC (tet‐on zmPDC/H113Q‐GFP‐V5/His) following doxycycline‐induction of enzyme expression. The bicarbonate/pyruvate signal ratio in untreated xenografts was 3.1 × 10−2 ± 3.1 × 10−2  and 2.3 × 10−2  after 96 h of doxycycline treatment. There was also no change when the bicarbonate signal was expressed relative to the total observable 13C signal, which was 6.0 × 10−3 ± 3.0 × 10−3 [±SD], n = 3) prior to treatment and 6.0 × 10−3  after 96 h of doxycycline treatment.Xenografts derived from cells transfected with 13C] acetaldehyde from [2‐13C] pyruvate. However, a [1‐13C] acetaldehyde resonance was not detectable in experiments using hyperpolarized [2‐13C] pyruvate in suspensions of PDC‐expressing cells (data not shown). In experiments on xenografts derived from these cells, a resonance at 209.4 ppm, corresponding to the resonance frequency of [1‐13C] acetaldehyde, was observed in some summed 1H ‐decoupled 13C spectra 96 h after doxycycline administration (five out of 10 experiments)  1H ‐decoupled spectra acquired from xenografts, both before and after doxycycline administration  at 96 h after doxycycline administration.Because in many tissues there will be a background bicarbonate signal, we also investigated whether PDC activity could be assessed from the production of [1‐zmPDC‐GFP‐V5/His fusion protein can be detected in vivo using hyperpolarized [1‐13C] pyruvate and 13C MRS and therefore that this system has the potential to be used as an MR gene reporter. The size of the zmPDC coding sequence (1.60 kbp), although considerably larger than that of GFP (0.73 kbp) and similar fluorescent reporter genes, is comparable to that of the widely used bioluminescence reporter, Firefly luciferase (1.65 kbp). A compact reporter size is an advantage when the payload constraints of delivery vectors are considered. Because it is a spectroscopic method in principle, it could be used with multiple gene reporters; for example, we have shown recently that the urea transporter can be used as a gene reporter in conjunction with hyperpolarized [13C]urea 13C resonances of urea, [1‐13C] pyruvate, and bicarbonate are resolved, therefore both reporters could be used in the same system. This is in general not possible with MRI methods that employ contrast agents modulating T1 or T2/T2* relaxation times, nor for positron emission tomography–based reporter genes We have demonstrated that the pyruvate decarboxylase activity expressed by the olarized ‐13C pyruzmPDC‐GFP‐V5/His fusion protein caused a decrease in cell growth rate in vitro , at least in HEK293T cells.Cell lysis increased the rate of  pyruvate 13C] pyruvate The background hyperpolarized 2 by mitochondrial PDH activity. However, this is unlikely, because doxycycline had no effect on CO2 production in wild‐type cells, where the bicarbonate/pyruvate signal ratio was unaffected by doxycycline addition and was similar to the ratio observed in untreated cells transfected with the inducible wild‐type PDC vector  as well as its close proximity to the much larger pyruvate signal. In addition, rapid metabolic conversion of [1‐13C] acetaldehyde to [1‐13C] acetate, and further to [1‐13C] AcCoA and [1‐13C] acetyl‐carnitine, has been observed previously in experiments with hyperpolarized [1‐13C] acetate in vivo 13C label flux between hyperpolarized  ethanol and [1‐13C] acetate in mouse liver showed no evidence of the  acetaldehyde intermediate, despite the much longer T1 of the deuterated acetaldehyde, which was approximately 19 s. Again this was attributed to a low steady‐state concentration of the labeled intermediate 13C] acetaldehyde exists in equilibrium with the hydrated form, with the hydrate 13C resonance at 91.4 ppm (data not shown), this will further decrease the sensitivity of its detection by 13C MRS 13C] pyruvate is likely to be from [1‐13C] AcCoA produced by PDH activity, as has been observed previously in heart muscle following administration of hyperpolarized [2‐13C] pyruvate 13C] acetate formation resulting from aldehyde dehydrogenase 2–catalyzed oxidation of the [1‐13C] acetaldehyde produced by PDC and that this is then rapidly converted to AcCoA by thiokinase. Build‐up of AcCoA has been observed in alcoholic fatty liver disease, where acetate produced by mitochondrial aldehyde dehydrogenase 2 from ethanol‐derived acetaldehyde is converted to AcCoA, but its further metabolism via the citric acid cycle is limited due to product inhibition of isocitrate dehydrogenase and α‐ketoglutarate dehydrogenase by NADH produced during acetaldehyde oxidation An alternative approach to circumvent the problem of background bicarbonate signal would be to use hyperpolarized [2‐zmPDC as an MR gene reporter when used with hyperpolarized [1‐13C] pyruvate. However, the levels of bicarbonate produced above background were low, which would make it challenging to use in conjunction with an imaging experiment. Increasing zmPDC expression, at least in the cells used here, is unlikely to improve the sensitivity of the reporter, because PDC activity appeared to be limited by pyruvate transport into the cell. However, the potential for increased temporal resolution resulting from rapid signal acquisition coupled with subsequent rapid decay of the hyperpolarized signals and the capability of this reporter to be combined with other spectroscopy‐based gene reporters in the same experiment merits further development of this and other enzyme‐based gene reporters for use with hyperpolarized 13C MRS.In conclusion, we have demonstrated the feasibility of using Supporting Figure S1. 1H STEAM spectrum acquired from a 1 cm3 voxel within the xenograft after intraperitoneal administration of [U‐2H3] pyruvate. The animals were given 10 mg/mL doxycycline in their drinking water for 72 h prior to the experiments. The inset shows an expansion of the 10–8 ppm region of the spectrum with the vertical scale expanded by 10. The spectrum is representative of two independent experiments. The assignments were made by comparison with published spectra .Click here for additional data file.


When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X‐ray data show how Tyr34 maintains solvent exclusion and the core H‐bond network in the active site by relocating to replace the missing Arg85′ sidechain. The 19F NMR data show deshielding effects that indicate the main function of Arg85′ is electronic polarization of the transferring phosphoryl group, primarily mediated by H‐bonding to O3G and thence to PG. DFT calculations identify electron‐density redistribution and pinpoint why the TS for guanosine 5′‐triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85′Ala relative to wild‐type (WT) RhoGAP. This study demonstrates that 19F NMR measurements, in combination with X‐ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site‐specific modifications.We report X‐ray crystallographic and  These studies show that the sidechain of a highly conserved arginine (Arg85′ in RhoGAP), termed the “arginine finger”, is inserted into the active site of RhoA in the RhoA/RhoGAP/GTP complex. Four functions have been proposed for the arginine finger in catalysis.3− transfer.The turnover number of GTP hydrolysis by RhoA is accelerated 3.9×103−, and the Arg85′Ala mutant RhoGAP (Arg85′Ala complex) was determined to 2.4 Å using molecular replacement , and showed the same perturbation of Switch I . Switch I is at the interface between RhoA and RhoGAP, and remote from the packing surface with neighboring molecules in the unit cell, thus showing that this perturbed conformation is not a crystallographic artefact. Indeed, this conformation is similar to that reported in the Cdc42/Cdc42GAPArg305′Ala/GDP/AlF3 TSA complex (PDB ID: 2ngr).We now report the integrated use of X‐ray crystallography, NMR, and DFT methods to determine changes in TS properties following site‐directed mutagenesis of Arg85′ to Ala in RhoGAP, and hence to dissect the molecular interactions underlying the deceleration of GTP hydrolysis in the RhoA/RhoGAP complex when the arginine finger is deleted. The X‐ray structure of a TSA complex containing GDP, MgF19F NMR, given the ability of 19F chemical shifts to report indirectly on the electronic environment for PO3− groups in TSs.4− complex  due to fast exchange of the four fluorines between the three binding sites for the oxygens of PO3−.4− .The two Arg85′Ala TSA complexes of RhoA were examined by 3− complex exhibits three well‐resolved 19F resonances   were therefore used to obtain a preliminary assignment. After optimization, the H‐bond distance from Tyr34‐OH to F3 in the model relaxed from 2.4 Å in PDB: 5m6x to 2.58 Å, and 19F NMR chemical shifts were calculated  using standard methods (see the Supporting Information).The MgFs Figure . The ass19F signals. Thus, SIIS values for both WT and Arg85′Ala MgF3− complexes were measured in mixed D2O/H2O solutions , while the resonance of F2 moves downfield by 3.5 ppm. The SIIS values for both F2 and F3 marginally decrease (by 0.2 ppm) compared to WT, thus implying that MgF3− is coordinated less tightly in the mutant complex than in the WT complex.A partial deuteration strategy experimentally confirmed the DFT‐based assignment of both downfield 19F NMR data indicate that removal of the arginine finger gives rise to a localized electron‐density redistribution within the TSA complex.3− TS, and thereby assess how the mutation modifies the TS structure. This was accomplished using an optimized computational model of the TS in the RhoA/RhoGAPArg85′Ala/GTP complex . One unique vibrational mode characterizing the motion of PG along the reaction coordinate confirmed that this structure corresponded to a TS (Movie S1). The principal features of the computed TS are “in‐line” trigonal bipyramidal geometry (178.2°), a 4.29 Å apical separation of donor and acceptor oxygens, and a relatively short H‐bond between Tyr34‐OH and O3G  distance of 1.99 Å, and a PG−O3B (leaving group oxygen of GDP) bond length of 2.30 Å . A similar non‐symmetry is observed in the TSA complex for MgF3− in PDB: 5m6x . Electron‐density surface maps provide interesting insight into the computational WT and Arg85′Ala transition states. Thus, when contoured at 0.304 e− Å−3, the surface density between O3B, PG and Ow3 is continuous in the WT TS whereas it is discontinuous between O3B and PG in the Arg85′Ala TS . These suggest that there is a corresponding increase in electron density only on O3G in the Arg85′Ala TS, which corresponds to the changes in 19F NMR chemical shifts in the TSA complexes. The marked upfield shift of F3 on changing one of its H‐bond partners from Arg85′ to Tyr34 therefore results from significant deshielding by Arg85′ in the WT TSA complex.Notwithstanding these differences in electron density, the w3 water hydrogens form regular hydrogen bonds to Thr37 and Gln63 in both computed TSs G in the absence of a H‐bond from Arg85′. Moreover, replacing three H‐bonds from Arg85′ by one involving Tyr34 maintains the H‐bond network identified in our earlier study of the WT complex.Based on the computed bond lengths and T Figure  indicate3G is the major factor in raising the TS energy for phosphoryl group transfer in the Arg85′Ala complex. Our work therefore supports previous QM/MM studies of the cognate mutation in the Ras/RasGAP complex.G by the formation of a stronger H‐bond to O3G.We conclude that loss of the positive charge from Arg85′ and the corresponding impact on its H‐bonding to O19F NMR measurements, in combination with DFT calculations and X‐ray crystal structures, offer a direct experimental method for dissecting the responses of a phosphoryl transfer enzyme to site‐directed residue modification, and can reliably identify even relatively small changes in TS properties. It demonstrates the value of using high‐level QM calculations to interpret 19F NMR chemical‐shift changes arising from the effect of site‐specific mutations on the complicated electrostatic environments present in enzyme active sites. These methods also deliver a complete 3D map of electron redistribution in the active site resulting from the mutation. The general strategy reported here opens up the possibility of employing 19F NMR measurements to assess the impact of naturally occurring, and in many cases disease‐causing, mutations on the ability of GTPases to catalyze phosphoryl transfer.Overall, this study shows that 19F NMR, and DFT computations were as described previously,Experimental methods for X‐ray crystallography, The 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.SupplementaryClick here for additional data file.


The structure of the glass is examined by X-ray diffraction and Raman spectra. The dynamic parameters such as the fragility, stretching exponent and non-linear factor are determined. A low fragility of m = 27 is exhibited for the chalcogenide, however, the stretching exponent is found not to have a larger value. The enthalpy relaxation spectra are constructed for various glass formers, and a relationship between the fragility and the symmetry of the spectra is demonstrated. The dynamic results are used to evaluate the structure of the Ge22Se78 glass.The enthalpy relaxation is performed in the glassy Ge Based on the viscosity data of the GexSex100− melts, the fragility in the compositions of 10 < x < 25 is calculated to range from 22.5 to 32458Se92 and Ge12Se88 reported m = 58–59xSex100− alloys with 10 < x < 33, the fragility m index was reported to be 14.8–29 using the modulated differential scanning calorimetry (mDSC)xSex1− (x ≤ 15) presented a continuous decrease in fragility from 68 to 28 measured with a constant ratio between the cooling and subsequent heating ratesβKWW values (or the non-linear factor x) determined in the enthalpy relaxation measurements are not consistent. It is also found that the values of βKWW and x determined by the enthalpy relaxation are notably higher than those determined by the volume relaxationConsiderable efforts have been made to explore the dynamics in the Ge22Se78 chalcogenide glass is studied, referring to earlier studies in GexSex1− where a minimum fragility is identified to fall in the compositions of 19 < x < 26, preferentially in the range of 21.5 < x < 2345m, βKWW and x are determined using the enthalpy relaxation, because the method has proven to be capable of producing quite comparable results with the viscosity or dielectric-relaxation measurements2322Se78 glass is determined to be low with m = 27. Surprisingly, a low stretching exponent is also revealed. The dynamic results are used to understand the structure of the Ge22Se78 glass.Given accurate dynamic parameters are crucial to understand the relaxation behaviors and the structure in glassy materials1222Se78 samples synthesized for 24 + 10 hours (details are presented in the Sample preparation section) and 24 + 168 hours. The nature of completely amorphous states is identified for the two samples by the XRD patterns using the wave vector gauged by q = 4π sinθ/λ. The intermediate-range order is unambiguously observed by the featured first-sharp-diffraction-peak (FSDP) at q = 1.09 Å−1, given that the FSDPs in many non-metallic network glassy materials are typically located at q ~ 1–2 Å−1 −1 along with a shoulder located at ~210 cm−1 and (b) a broad band centered at 259 cm−1 with high-intensity, spanning from 225 to 280 cm−1. The 195 cm−1 band in region (a) is assigned specifically to the breathing mode of corner-shared (CS) GeSe4 tetrahedral units, and the 210 cm−1 band is assigned to the vibration of Se atoms involved in edge shared (ES) GeSe4 tetrahedra26−1 is characteristic of pure Se and can be assigned to Se-Se stretching modes (SM) in (Se)n units2822Se78 samples synthesized for 24 + 10 hours and 24 + 168 hours before quenching. Comparing the curves in the two panels, it is seen that the onset glass transition temperature of the sample synthesized for 24 + 168 hours is a bit lower. The heatflow curves are recorded at a fixed heating rate of 20 K/min after the glasses are quenched at different cooling rates of 20, 10, 5, 2.5 and 1 K/min, from which the fictive temperatures are determined. The fictive temperature, Tf, firstly proposed by Tool23Tf of glasses can be calculated from the enthalpy-matching method for the heating curves using Moynihan’s construction, following the relation, T* is an arbitrary temperature above Tg, Cp-liquid and Cp-glass denote the heat capacity of liquid and glassy states, respectively. Principally, Tf of each glass quenched at a specific cooling rate can be independently calculated from the heating curve. In order to improve the accuracy, we chose the enthalpy-compensation method, H is the energy difference between the glass denoted by Tf and the reference glass by Cp difference between the two glasses, ΔCp is the Cp difference between the liquid and the glass at Tg, which is shown in the inset of Fig. 3(a)Tf on the cooling rate Q for the two glassy samples subjected to distinct synthesis time in melts. Using the expression of ln Q = A − Eg/RTf, where R is the gas constant, A a constant, and Eg the apparent glass transition activation energym-index is determined to be 23 ± 2 for the glass synthesized for 24 + 10 hours and 27 ± 2 for the 24 + 168 hours in terms of m = Eg/2.303RTg32xSex1− systems are also reproduced in 45m = 24 at x = 0.225. The fragility determined in the present work are largely comparable with the reported results based on different methods such as viscosity45Cp curves of the Ge22Se78 glass with synthesis time of 24 + 168 hours are shown in Cp curves, are presented in 34Tg22Se78 glass is shown in HR to be 0.708 J/g.The cooling/heating capacity Cp curves for a group of glass forming liquids of diverse fragility with temperature normalized to their S = F2/F1 to reflect the symmetry of the spectra as shown in the inset of F1 and F2 denote the half width at half maximum of the peak in the left and right flanks, respectively, and therefore, F1 + F2 defines the full width at half maximum. S and fragility. The majority of the data are seen to fall into a master curve, and increased symmetry of the relaxation spectra is evident for strong liquids. Based on the correlation shown in mmin = 16.To understand the enthalpy relaxation spectra in temperature domain defined by the enthalpy difference curves, we define a parameter of Cp curves of the glasses quenched at a cooling rate of 20 K/min are analyzed using the Tool–Narayanaswamy–Moynihan–Hodge (TNMH) equations, which emphasize the effects of fragility, nonlinear parameter x and stretching exponent βKWW24Cp curves and the temperature derivative of the fictive temperature, dTf /dT , apparent activation energy (Ea = ln10RTgm), x and βKWW. With m = 27 available for the Ge22Se78 glass, the fit gives the two parameters, βKWW and x, to be 0.43 ± 0.05 and 0.64 ± 0.05 respectively as shown in Tg and the marked glass-transition heat capacity increment for the Ge22Se78 glass are advantageous for the enhanced accuracy in the DSC measurements and, consequently guarantee the reliable results of the non-exponential and non-linear parameters involved in the enthalpy relaxation. It is a surprise to see such a low βKWWvalue for the Ge22Se78 glass presents the large deviation from the general correlation between m and βKWWThe heating (upscan) 22Se78 glass indicates that the glass former belongs to the category of strong dynamics according to the Angell’s strong-fragile classification schemem-index spans from the hitherto strongest SiO2 of m = 20 to the most fragile cis/trans-decalin of m = 145m = 27) for the Ge22Se78 alloy is somehow unexpected since, for inorganic substances, the extremely low fragility is generally associated with the network glass-forming materials with the MX2 stoichiometry such as SiO2 and GeO2, where the structures are built predominantly by tetrahedral MX4 motifs12243840414222Se78 fragility is quite comparable with those of BeF2 (m = 24) and ZnCl2 (m = 30) glasses41The determination of the fragility for the Gem indexes generally have relatively high βKWW values 14βKWW of 0.43 is unusual when compared with the strong Ge22Se78 glass of m = 27. In the earlier studies of the βKWW data produced by enthalpy relaxation and dynamic measurements such as dielectric and mechanical relaxations for various glass formers, excellent consistency is often exhibited2438βKWW value of 0.43 makes the Ge22Se78 glass to be an exception in the strong glass formers with simple compositions14Studies found that strong glass formers with low βKWW values than those of pure components, suggesting effective broadening of the relaxation dispersion47βKWW values are evident22Se78 glass, it is, consequently, speculated that the low fragility m-index and the small βKWW value might be partly associated with the mixing effect. This is quantitatively consistent with the random connectivity of the basic structure motifs of GeSe4 tetrahedra and (Se)n units in the Ge22Se78 glass, as involved in the reported models6292 (much different from those of GeO2 or SiO2)5122Se78 glass78929Dynamic behaviors in glass formers have been recognized to be associated with the structural features of glasses and melts. Our recent studies of binary glass forming mixtures47x > βKWW for the Ge22Se78 glass. This relation is quite rarely reported for most of glass formers, where x is generally smaller than βKWW2446.75Ti8.25Cu7.5Ni10Be27.515Te855649Cu26.9Ag5.5Pd2.3Si16.3The present studies also detect a relation of 22Se78 was prepared with the melt-quenching method−3 Pa. The mixtures were heated up to 1230 K, higher than The sample glass Geanalyses .α, λ = 1.54 Å) and Raman measurements on a Renishaw inVia micro Raman spectroscopy with a laser radiation of 514 nm. Heating and cooling heat capacity curves were obtained by using a differential scanning calorimeter  calibrated by using indium and zinc as references. The sample mass varied from 6 to 10 mg. The calorimetric glass transition temperature Tg was defined as the onset temperature of the heat capacity jump from the glassy to the liquid state. Enthalpy relaxation was performed in two manners. One used a cooling/heating cycle at the same rates of 20 K/min, and the enthalpy difference between the cooling and subsequent heating defines the relaxation enthalpy. The other is based on the identification of the enthalpy difference in the glasses quenched by various cooling rates, of which the enthalpy difference is calculated from the integral of the heating Cp difference among the glasses quenched at various cooling rates but fixed heating rate, typically 20 K/min. Usually, the heat capacity curve generated from cooling/heating rates of 20 K/min is set as the reference. This method has been described elsewhereCp measurement temperature spans from Tg − 100 K to Tg + 50 K, which can guarantee that no relaxation occurs at low temperature regimes, and equilibrium (supercooled liquids) is reached at high temperature regimes.The structure of the melt-quenched samples were checked via the x-ray power diffraction in a Rigaku D/MAX/2500/PC .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The spatial distribution of DSB repair factors γH2AX, 53BP1 and Rad51 in ionizing radiation induced foci (IRIF) in HeLa cells using super resolution STED nanoscopy after low and high linear energy transfer (LET) irradiation was investigated. 53BP1 and γH2AX form IRIF with same mean size of (540 ± 40) nm after high LET irradiation while the size after low LET irradiation is significantly smaller. The IRIF of both repair factors show nanostructures with partial anti-correlation. These structures are related to domains formed within the chromatin territories marked by γH2AX while 53BP1 is mainly situated in the perichromatin region. The nanostructures have a mean size of (129 ± 6) nm and are found to be irrespective of the applied LET and the labelled damage marker. In contrast, Rad51 shows no nanostructure and a mean size of (143 ± 13) nm independent of LET. Although Rad51 is surrounded by 53BP1 it strongly anti-correlates meaning an exclusion of 53BP1 next to DSB when decision for homologous DSB repair happened. Ionizing radiation induces a variety of different types of damage when targeted to living cells. Severe damage, which can influence cell survival or lead to carcinogenesis, occurs due to ionizing events in the DNA molecule itself. The most lethal of these types of DNA damages are the double-strand breaks (DSB), as they may lead to genetic alterations which in turn can be responsible for cell death or carcigonesis. Mammalian cells react with a variety of complex response mechanisms to DSB induction. One main reaction is the phosphorylation of the histone variant H2AX at serine 139 (S139) to obtain γH2AX through kinases such as ATM, ATR and DNA-PK234523Recent work analyzed the clustering of DSB repair factors in detail using high resolution microscopy10111113141523513151718et al.212223Bekker-Jensen It is well known that the number and complexity of DSB are larger when induced by high linear energy transfer (LET) radiation compared to low LET radiation of the same dose1128Measurements of high LET radiation and the Rad51-53BP1 experiment for low LET proton radiation were performed in 3 different experiments with two repetitions  with a nIRIF induced by irradiation of human HeLa cells with (27 ± 8) MeV carbon ions resulting in an LET value of (500 ± 80) keV/μm and with (20.8 ± 0.1) MeV protons (LET = (2.6 ± 1) keV/μm) were examined. Visualization of IRIF from 53BP1, γH2AX and Rad51 was performed by 3D STED nanoscopy using secondary immunofluorescence staining after 1 h of post-irradiation incubation. This super resolution optical microscopy method provided a resolution of 105 nm in lateral direction and approximately 200 nm in axial direction in this experiment. 1330The reduced product of the differences from the mean (rPDM)et al.By contrast, the rPDM analysis for 53BP1 and Rad51 mainly shows anticorrelation cf.  V, repreet al.x = 0 and Δy = 0 the correlation has a maximum with Pearson coefficient of 1. The larger the shift in both directions the smaller the correlation becomes, but the decrease depends on the mean structure size in the image. The determined curves for x-shift with Δy = 0 and y-shift with Δx = 0 reflect the mean structure sizes as shown for an ACF-evaluation of an IRIF of a carbon irradiated cell for 53BP1 in σ the measured width and σ1 the actual width in the image. This is true for every structure of different size occurring in the image. The two widths of the two Gaussian functions fitted to the ACF distribution for 53BP1 and γH2AX IRIF can be interpreted as the full widths at half maximum (fwhm) of the nanostructure and the gross structure each multiplied by a factor of The size of structures repeatedly occurring in the microscope images are measured using the auto correlation function (ACF). The ACF was determined using the approach introduced by Van Steensel The size measurements were done in two different labelling setups  for 2 irradiation modes on 170 IRIF altogether. The ACF analysis of γH2AX and 53BP1 show a superposition of two Gaussian in both irradiation cases, low and high LET.The obtained full width at half maximum (fwhm) values of the IRIF are presented in Super resolution STED nanoscopy was used to identify the structural relationships occurring in ionizing radiation induced 53BP1, γH2AX and Rad51 IRIF in human HeLa cells for low and high LET irradiation. The correlating and anticorrelating areas of IRIF from different repair factors were quantitatively analysed using the rPDM approach. By introducing the autocorrelation function as modified from the Van Steensel approachThe microscopic structures of 53BP1 and γH2AX IRIF look quite similar: they have a similar total IRIF size of (540 ± 40) nm fwhm for carbon ion induced foci and also a same but significantly smaller IRIF size for low LET proton irradiation (412 ± 21) nm). They also show a similar nanostructure size of about 130 nm being also the same for both, low and high LET radiation. The nanostructure size is larger than the microscope resolution (105 nm) and therefore it is concluded that this is true size of the clustered proteins. On a micrometer scale, the two repair factors correlate and due to their large IRIF sizes the conclusion can be drawn that 53BP1 and γH2AX are both part of the flanking chromatin DSB repair compartment as it has been described before13 nm). The2223Complementary to the 53BP1 and γH2AX, the Rad51 IRIF are represented by a single structure of total size 135 ± 10) nm without any nanostructure. The Rad51 IRIF is localized in the single-stranded DNA (ssDNA) compartment5 ± 10 nmet al.In this study we used the approach of correlating the nanostructures of different repair factors to each other to get deeper insight in the higher order chromatin structure. Although no chromatin labelling was performed the results are in good correspondence to findings of former studies, where e.g. 53BP1 was found not to penetrate dense chromatin domains2) in RPMI medium (Sigma Aldrich), which was supplemented with 10% FCS and 1% Penicillin/Streptomycin. Cells were seeded on 22 × 22 mm2 coverslips several hours prior to irradiation, so that at the time of irradiation the cells were in the exponential growth phase. Irradiation was performed at the microirradiation facility SNAKEμm thick medium layer, which was determined by weight measurement of the sample with medium and totally dried out. The thickness was calculated as A = 22 × 22 mm2 the area of the coverslip and the density of water μm for the medium layer. Irradiation was performed in a field of 3.5 × 22 mm2 under an angle of 9° with respect to the cell layer and lasted a few seconds. The fluence of the carbon ion beam was HeLa cells  were cultivated at 37 °C  para-formaldehyde solution, washed with phosphate-buffered saline (PBS), and permeabilized with a 0.1% (v/v) Triton-X-100 solution. Then the cells were blocked with PBS containing 1% bovine serum albumin (BSA) and 0.15% glycine, as previously describedA super resolution optical CW STED microscope (Leica TCS SP 8 3X) was used to obtain the detailed structures of the IRIF. For the Abberior STAR 440SX an excitation laser wavelength of 470 nm and for the Chromeo505 a wavelength of 514 nm were used. The detection ranges were 473 nm to 504 nm and 518 nm to 580 nm respectively, with a depletion laser at 592 nm. Laser power for the excitation laser is in the range of 1 mW and for the STED laser around 70 mW. The STED laser was subdivided in a lateral STED beam (40% of the energy) and an axial STED beam (60%). The system was aligned with regard to temporal and temperature dependent shift as described beforeσ = 50nm for the nanostructure and approx. σ = 180 nm. The sample size is calculated as The measurements were performed in 3 different experiments which were carried out twice as displayed in et al.For correlation analysis, the rPDM method as described by Reindl is calculated. The value ranges from −1 for anticorrelation to +1 for correlation. A 2D Correlation map is formed where the individual pixel correlation coefficient is reprinted as an image.et al.The samples were analyzed using cross correlation functions (CCF) as introduced by Van Steensel Ai, Bi the gray values for the pixel i in the first and second image, respectively. Amean, Bmean are the mean gray values for each image. The CCF is then obtained by plotting the Pearson coefficient against the shift Δx. For the determination of the structure sizes of an IRIF originating from one damage marker, i.e. γH2AX, 53BP1 or Rad51, it is necessary to obtain the so called autocorrelation function (ACF), which is the CCF for an image correlated with itself. In this study, the shifts were performed in x- as well as in y-direction, which creates a shifting matrix where the columns represent the x-shift, the rows the y-shift and the entries are the Pearson correlation coefficient r for the corresponding x- and y-shift. By plotting the Pearson coefficient against both x- and y-shift, a 2D CCF or ACF can be determined. For the quantitative analysis the projection of the 2D CCF/ACF both for x- shift (Δy = 0) and for y-shift (Δy = 0) are used.with The ACF is a symmetrical correlation problem and therefore the ACF reduces to a convolution problemIn an image, which has several structures, like micrometer and nanostructure, the structure can be represented by a sum of several Gaussian functions. Therefore, for determining the structure sizes, a superposition of several Gaussian functions was fitted to the projection of the 2D ACF. The resulting full width at half maximum (fwhm) of each Gaussian represents one underlying structure size multiplied by the factor In the case of the damage markers used in this study, one or two structure sizes are visible, respectively, the size of the IRIF and the nanostructure size. They are determined using a superposition of two Gaussian functions:y0 is the mean Pearson correlation coefficient for large shifts, A is the amplitude of one of the two Gaussian functions and, as the maximum occurring value is 1, the height of the second Gaussian is (1−A−y0)). σ1 and σ2 are the standard deviations of the two Gaussian functions. Using the standard deviations the fwhm can be calculated to fwhm2 = 2.35 × σ2 and fwhm1 = 2.35 × σ1.1 representing the size of the IRIF. The second Gaussian function describes the smaller structure occurring inside an IRIF with a size of fwhm2. For Rad51 the second structure size drops to 0, because no visible nanostructure occurred and only the size determined by fwhm1 remains.The sum of the Gaussians f(x) is suitable for the ACF either for γH2AX or 53BP1 where the first Gaussian function describes the IRIF macrostructure with fwhmHow to cite this article: Reindl, J. et al. Chromatin organization revealed by nanostructure of irradiation induced γH2AX, 53BP1 and Rad51 foci. Sci. Rep.7, 40616; doi: 10.1038/srep40616 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Arabidopsis thaliana, AtGSTF2, revealed by X‐ray crystallography. Complexes of the AtGSTF2 dimer were obtained with indole‐3‐aldehyde, camalexin, the flavonoid quercetrin and its non‐rhamnosylated analogue quercetin, at resolutions of 2.00, 2.77, 2.25 and 2.38 Å respectively. Two symmetry‐equivalent‐binding sites (L1) were identified at the periphery of the dimer, and one more (L2) at the dimer interface. In the complexes, indole‐3‐aldehyde and quercetrin were found at both L1 and L2 sites, but camalexin was found only at the L1 sites and quercetin only at the L2 site. Ligand binding at each site appeared to be largely determined through hydrophobic interactions. The crystallographic studies support previous conclusions made on ligand binding in noncatalytic sites by AtGSTF2 based on isothermal calorimetry experiments  Biochem J 438, 63–70) and suggest a mode of ligand binding in GSTs commensurate with a possible role in ligand transport.Glutathione transferases (GSTs) are involved in many processes in plant biochemistry, with their best characterised role being the detoxification of xenobiotics through their conjugation with glutathione. GSTs have also been implicated in noncatalytic roles, including the binding and transport of small heterocyclic ligands such as indole hormones, phytoalexins and flavonoids. Although evidence for ligand binding and transport has been obtained using gene deletions and ligand binding studies on purified GSTs, there has been no structural evidence for the binding of relevant ligands in noncatalytic sites. Here we provide evidence of noncatalytic ligand‐binding sites in the phi class GST from the model plant  CDNB1‐chloro‐2,4‐dinitrobenzeneDMSOdimethyl sulfoxideGSTglutathione transferaseITCisothermal calorimetryLBLuria–BertaniPDBProtein Data BankPEGpolyethylene glycolE.C. 2.5.1.18) are a large group of enzymes with a major role in the detoxification of xenobiotic compounds Ka from 8.7 to 6.2, so as to form a thiolate of high nucleophilic reactivity Arabidopsis thaliana (At), a model species for plant genetic studies, 54 soluble GSTs plus one membrane‐associated protein in eicosanoid and glutathione metabolism have been identified AtGSTZ1 has been shown to have identical roles to its human homologue (HsGSTZ1) with respect to tyrosine and phenylalanine catabolism Glutathione transferases  O‐glucoside with GSH. However, glutathionylated anthocyanins have not been identified in plant cells. Furthermore, a phi class GST from Petunia, named AN9, was also shown to be involved in anthocyanin biosynthesis, but this was not dependent on conjugating activity towards these pigments in vitroArabidopsis AtGSTF12 (TT19) have shown that the protein can directly bind cyanidin and cyanidin‐3‐O‐glucoside Paradoxically, while GSTs have a conserved ability to bind GSH, the only clearly established role for GSTs demonstrated Arabidopsis phi class AtGSTF2 has been the subject of several ligand binding studies, following the observation that the protein bound both indole‐3‐acetic acid (IAA) and 1‐N‐naphthylpthalamic acid (NPA), an endogenous flavonoid regulator of auxin transport AtGSTF2. Later studies showed that purified recombinant AtGSTF2 bound a range of heterocyclic compounds, including the flavonoid quercetrin (quercetin‐3‐O‐rhamnoside), the indoles camalexin, harmane, norharmane and indole‐3‐aldehyde and the flavin lumichrome Schistosoma japonicaS‐(4‐nitrophenacyl)glutathione was bound, was found buried more deeply within the dimer interface S‐(p‐nitrobenzyl)‐glutathione, was reported to bind to a peripheral hydrophobic binding site in the tau class GST GmGSTU4‐4 from Glycine maxZea mays (ZmGSTF1) that overlapped with the ‘G’‐ and ‘H’‐site in vitro evidence for small molecule binding by members of the phi class of GSTs in Arabidopsis, few other structural insights into these interactions have yet been reported. In order to obtain further insight into the ligand transport properties of plant GSTs, we now report X‐ray crystallographic studies conducted with AtGSTF2 in the presence of a range of ligands. Three structurally distinct ligands out of six of those identified as binding partners for AtGSTF2 in previous studies were selected for study, namely indole‐3‐aldehyde 1, camalexin 2 and quercetrin 3  studies previously reported AtGSTF2, knowledge of which will be important in understanding the involvement of these proteins in the binding and transport of small molecules in various plant physiological processes.The AtGSTF2 gene, as prepared by Dixon et al. Escherichia coli Tuner (DE3) cells  that also contained the pRARE plasmid from Rosetta (Merck‐Millipore). Transformants were grown on Luria–Bertani (LB) agar supplemented with 100 μg·mL−1 of kanamycin and 50 μg·mL−1 of chloramphenicol at 37 °C. A single colony of a plate grown overnight was used to inoculate 4 × 5 mL of LB broth. These starter cultures were grown overnight at 37 °C with shaking at 180 r.p.m. and were then used to inoculate LB broth (4 × 500 mL cultures) in which cells were grown until the optical density (OD600) of the culture had reached approximately 0.6. At this point, the expression of AtGSTF2 was induced by the addition of isopropyl β‐D‐1‐thiogalactopyranoside . The cultures were then incubated at 20 °C in an orbital shaker overnight at 180 r.p.m. After approximately 18‐h growth, the cells in each case were harvested by centrifugation at 4225 g for 15 min in a Sorvall RC5B Plus centrifuge  and were then resuspended in Tris/HCl buffer pH 7.5 . Cells were disrupted by ultrasonication for 3 × 30 s bursts at 4 °C with 1‐min intervals, and the soluble and insoluble material fractions were separated by centrifugation at 26 892 g for 30 min. The supernatant, containing the soluble AtGSTF2, was loaded onto a 10 mL GSH sepharose 4B . Column fractions were analysed by SDS/PAGE and the fractions containing purified proteins were pooled and concentrated using a 10 kDa cut‐off Centricon® filter membrane (Merck‐Millipore). Concentrated protein was loaded onto an S75 Superdex™ gel filtration column  that had been equilibrated with buffer also including addition of 150 mm NaCl. Fractions containing pure protein were pooled and stored at −20 °C.The pET24b vector containing the 1–4 were purchased from Sigma Aldrich . Pure AtGSTF2 was preincubated with ligands 1, 2, 3 or 4 prior to crystallisation experiments, at concentrations of either 5 or 10 mm for 1 h, followed by microcentrifugation at 16 300 g to remove any insoluble precipitates resulting from complexation. Ligand‐complexed proteins were then subjected to crystallisation trials using a Mosquito® ROBOT  and a range of commercially available crystallisation screens in 96‐well plate sitting drop format, in which each drop consisted of 150 nL protein and 150 of precipitant reservoir solution. Crystals of AtGSTF2 in complex with indole‐3‐aldehyde 1 and camalexin 2 were obtained in 0.2 m sodium acetate and 20% (w/v) polyethylene glycol 3350. Initial crystals observed for the complex of AtGSTF2 with quercetrin 3 and AtGSTF2 with quercetin 4 were in 0.1 m propanoic acid, cacodylate, bis‐tris propane system and 15% (w/v) polyethylene glycol 1.5K at pH 7.0. In all cases, a protein concentration of 10 mg·mL−1 was employed. Larger crystals for diffraction analysis were obtained using the hanging drop vapour diffusion method in 24‐well plate Linbro dishes, with 2 μL drops consisting of a 1 : 1 ratio of mother liquor to protein solution. The best crystals of AtGSTF2‐indole‐3‐aldehyde 1 and AtGSTF2‐camalexin 2 complexes were obtained in drops containing 0.2 m sodium acetate and 20% (w/v) polyethylene glycol 3350 with 1% (v/v) n‐propanol. For AtGSTF2‐quercetrin 3 and AtGSTF2‐quercetin 4 complexes, the best crystals were obtained from drops using the same conditions employed in the Mosquito® screen. Prior to analysis on in‐house X‐ray equipment, the crystals were washed with the mother liquor solution containing 20% (v/v) ethylene glycol as cryoprotectant and the appropriate ligand at the crystallisation concentration, followed by flash‐cooling in liquid nitrogen. Crystals were tested for diffraction using a Rigaku Micromax‐007HF fitted with Osmic multilayer optics  and a MARRESEARCH MAR345 imaging plate detector . Those crystals that diffracted to a resolution of equal to, or better than, 3 Å resolution were retained for data set collection at the synchrotron.Ligands 1 and 2 were collected on beamline I04‐1 and complexes with 3 and 4 on beamline I03. Data were processed and integrated using xdsscalaxia2 processing system AtGSTF2‐indole‐3‐aldehyde 1, AtGSTF2‐quercetrin 3 and AtGSTF2‐quercetin 4 were each in space group P212121, with six molecules in the asymmetric unit, constituting a trimer of dimers. The crystals of complex of AtGSTF2‐camalexin 2 were in space group P1 with 24 molecules in the asymmetric unit, consisting of four trimers of dimers. The structure of each complex was solved using molrepAtGSTF2  as the model. The solvent content in the AtGSTF2‐indole‐3‐aldehyde 1, AtGSTF2‐quercetrin 3 and AtGSTF2‐quercetin 4 complexes was 42% and in the AtGSTF2‐camalexin 2 complex was 47%. The structures were built and refined using iterative cycles using cootrefmacprodrg1 featured three molecules of 1 per dimer, with two at the L1 and one at the L2 sites. The complex with 2 featured two molecules per dimer, one at each of the L1 sites, and while there was some density at the L2 site, the occupancy was not considered sufficiently substantial to model the camalexin ligand. The complex with quercetrin 3 featured three ligands in the dimer, at L1 and L2, although, in two of the dimers, sufficient density was only observed for two ligands to be modelled. In addition, the rhamnose moiety of one of the seven ligands, at the L2 site between subunits ‘C’ and ‘D’, could not be modelled. The complex with quercetin 4 featured one ligand per dimer, at the L2 site in each case. The final structures exhibited % Rcryst and Rfree values of 19.9/23.4 (complex with 1); 25.0/28.4 (complex with 2); 21.4/25.2 (complex with 3); and 20.4/24.6 (complex with 4). All structures were finally validated upon deposition at the PDB. Refinement statistics for all structures are presented in Table 1 showed 98.4% of residues to be situated in the most favoured regions, 1.0% in additional allowed and 0.6% residues in outlier regions. For the complex with 2, the corresponding values were 96.5%, 3.0% and 0.5%. For the complex with 3, the corresponding values were 98.0%, 1.3% and 0.7%. For the complex with 4, the corresponding values were 98.2%, 1.0% and 0.8%. Coordinates and structure factors for AtGSTF2 complexes with indole‐3‐aldehyde 1, camalexin 2, quercetrin 3 and quercetin 4 have been deposited in the Protein Data Bank  with accession codes 5a4u, 5a5k, 5a4w and 5a4v respectively.Complete data sets described in this report were collected at Diamond Light Source; Didcot, Oxfordshire, UK. Complexes with AtGSTF2 has been published previously in complex with S‐hexylglutathione (1GNW) 1BX9) 1GNW, protomers of AtGSTF2 are found in a classical dimeric association. In order to determine crystallisation conditions for complex formation between AtGSTF2 and the heterocyclic ligands 1–4, fresh crystallisation screens were performed with the protein preincubated with 10 mm ligand (or 5 mm ligand in the case of the less soluble quercetin). Crystal complexes were obtained in each case.The crystal structure of 1GNWAtGSTF2 structures featured dimers in each ligand complex structure, with different numbers of monomers observed in the asymmetric unit depending on the space group. In the case of complexes formed with 1, 3 and 4, crystals grew in the P212121 space group, with six monomers in the asymmetric unit. In the case of camalexin 2, the space group was P1, with 24 monomers found. After building the peptide backbone, side chains and water molecules, clear residual density for each complex was observed in omit maps at an electron density level of 3σ that could be modelled as the relevant ligand in each case.The statistics for data collection and refinement are shown in Table AtGSTF2 dimer as observed in the complex with indole‐3‐aldehyde 1, with the selected alpha‐helices labelled for ease of reference. Secondary structure analysis on the PDB server (http://www.rcsb.org/pdb/) shows that each monomer contains 12 α‐helices , two 310 helices (36–38 and 40–42) and four β‐strands . For the complex with 1, representative monomer pairs in a dimer superimposed with an r.m.s.d. of 0.44 Å over 209 C‐alpha atoms, with no significant differences in amino acid side‐chain positions. For complexes with 2, 3 and 4, the r.m.s.d. values were 0.13, 0.37 and 0.35 Å respectively. The ligand 1 was observed in three locations in the dimer  and Phe52 and Phe66 (‘B’) of its neighbour. The plane of the aromatic ligand is sandwiched between the side chain of Arg154 and a hydrophobic shelf formed from (A)Ile102, (A)Val106 and (B)Phe52 and (B)Phe66. The aldehyde moiety is at a distance of 3.2 Å from the peptidic carbonyl of Leu161, 3.7 Å from the backbone carbonyl of Ile99, and 4.0 Å from the side‐chain hydroxyl of (A)Thr169. The indole nitrogen was not observed to make hydrogen bonding contact with any side chains in these sites.Detail of the ligand‐binding site 1 in the L2 site was almost entirely characterised by hydrophobic interactions His77, (A)Tyr97 and (B)Gln73, and the ligand was not successfully modelled here.The binding of indole‐3‐aldehyde ons Fig. B. The pl1, the plane of the camalexin ligand again lies within the hydrophobic pocket formed by the side chains of binding site L1 His77 and (B)Tyr97. The OAC atom of the resorcinol moiety is 3.5 Å from the side‐chain amide of (A)Gln73 and the OAF at a distance of 3.4 Å from the backbone carbonyl of (B)Ile94. The catechol ring is stacked between (A)Tyr97 and (B)His77. In contrast to quercetrin binding in the L1 site, the three rings of the flavone system are coplanar in the L2 site Ser91. The O2 hydroxyl of rhamnose is also 3.0 Å from the backbone carbonyl of (B)Lys92.Both quercetrin L1 and L2. These were remote from the active site of the enzyme more classically associated with interactions with xenobiotics, and their glutathionylated derivatives, formed following conjugation  1GWC) 1GWC, a tryptophan residue W101, which superimposes with Gly103 in AtGSTF2, occupies the L1 site. This Trp is also conserved in the phi GST F1 (4RI6) from poplar L2 site, the hydrophobic residues His77 and Trp97 that form the hydrophobic pocket binding the aromatic ligands are again not conserved in 1E6B or 1GWC, being replaced by Glu and Arg residues respectively, and Asp and Lys in 4RI6.The data reported herein represent the first structures of a plant GST complexed with natural products, through selective hydrophobic interactions localised to two newly identified ligand‐binding sites AtGSTF2 structures reported herein represent important types of biologically active plant secondary metabolites derived from indoles and polyphenols respectively. The selectivity of these binding interactions is suggestive of a physiological function. Roles associated with interactions located away from the active site are most likely related to sequestration and transport of biologically active ligands. A role for these interactions in the allosteric activation of AtGSTF2 has also been suggested, based on an observed increase in kcat for the conjugation of the model compound CDNB with GSH in the presence of harmane 1–4 in the present study (data not shown) and very little change in protein structure was observed when the ligand complexes obtained herein were compared with structures of the apo‐protein. While we recognise the possibilities of ligand binding as a crystallographic artefact at the ligand concentrations used in this study, both binding constants and enthalpy of complex formation values determined by ITC were presented by Dixon 3, which has the most polar functionality of ligands 1–4, binds in both L1 and L2 sites, and displays most hydrogen bonding interactions with the protein, gave a ΔH value of −21.1 kcal·mol−1 and a Ka of 0.16 μm−1 in that work. Indole‐3‐aldehyde 1, which also binds in both L1 and L2 sites, but makes fewer interactions, gave a less negative ΔH value of −13.7 kcal·mol−1 but a comparable value for the Ka, of 0.09 μm−1. Camalexin, which was observed to bind only in the L1 site, gave the least negative ΔH value of −9.3 kcal·mol−1, and a higher affinity constant of 0.84 μm−1.The classes of heterocyclic ligands bound within the AtGSTF2 being formally identified as an auxin‐binding protein The current study gives a structural basis for LA and ELR performed experiments; ELR, NCB, RE and GG designed experiments and supervised the work; RE and GG wrote the manuscript, with contributions from other authors.


R1 = 1/T1 of protons in H2O and HDO have never been measured in gas phase. We report R1 in gas phase in a field of 18.8 T, i.e., at a proton Larmor frequency ν0 = 800 MHz, at temperatures between 353 and 373 K and pressures between 9 and 101 kPa. By assuming that spin rotation is the dominant relaxation mechanism, we estimated the effective cross-section σJ for the transfer of angular momentum due to H2O-H2O and HDO-D2O collisions. Our results allow one to test theoretical predictions of the intermolecular potential of water in gas phase.In gas phase, collisions that affect the rotational angular momentum lead to the return of the magnetization to its equilibrium (relaxation) in Nuclear Magnetic Resonance (NMR). To the best of our knowledge, the longitudinal relaxation rates  Mz to its equilibrium after a perturbation, through a mechanism known as spin-rotation. If spin-rotation is the dominant mechanism, cross-sections for the transfer of angular momentum can be obtained from NMR relaxation rates in gas phase. Such relaxation rates have been measured over a wide range of pressures and temperatures89111214154, CH3D, CH2D2, and CHD3. In supercritical water1718para-water2021222324252728293031R1 = 1/T1 of gaseous H2O and HDO at different temperatures and pressures. To the best of our knowledge, this is the first time that such observations are reported. Our measurements are useful to refine intermolecular potentials for water vapour. These may be compared with water confined in matrices3321Water is the most extensively studied molecule on Earth. A precise determination of its intermolecular potential would allow accurate predictions of its properties in gas, liquid and solid phase. However, despite huge theoretical efforts13450, longitudinal NMR relaxation takes place. Comprehensive theoretical treatments of NMR relaxation can be found elsewhere3637383940414243Collisions between molecules can induce transitions between rotational quantum states. As a result, spin-dependent interactions vary as a function of time and, if the fluctuations occur at frequencies in the vicinity of the nuclear Larmor frequency ωSpin-rotation (SR) relaxation is due to collisions that modulate local fields induced at the sites of the nuclei by the rotation of the surrounding electronic cloud.Relaxation induced by spin-rotation can be described by ref. where:J is the spin-rotation correlation time, Ceff (in Hz) the spin-rotation constant, ωJ the rotational frequency (in rad/s)v is the average thermal velocity, σJ is the collisional cross-section for the transfer of angular momentum, I0 is the moment of inertia, grot is the g-factor, μN is the nuclear magneton, H is the magnetic field and μ is the reduced mass of the two colliding particles. The correlation time τJ is related to the lifetime of the rotational quantum states. The relaxation process can be described by characterizing the cross-section for the transfer of angular momentum. Intermolecular potentials used to model the interaction mostly consist of an isotropic part, usually a radial function, depending only on the distance between particles  and an anisotropic part, depending also on the orientation of the molecules with respect to each other. The intermolecular potential of a molecule can be written by considering its axial symmetryτC that is related to the mean time needed for the molecule to undergo a rotation through one radiant. DD relaxation can occur between spins in the same molecule (intramolecular DD) or between spins in different molecules (intermolecular DD). Relaxation by the intramolecular DD interaction between the two protons of water is described by ref. Dipole-dipole (DD) relaxation is due to fluctuations of the interaction between magnetic dipoles, which are induced by physical rotation. The DD interactions are described by a correlation time τr is the distance between the protons, γH is the gyromagnetic ratio of protons and μ0 is the magnetic permeability in vacuum.where C is linked to τJ by the Hubbard relationC/τJ = 1/6. For dilute gases (τJ → ∞), the ratio of correlation times τC/τJ varies from 5/4 (Ivanov model) to 1/4 (extended diffusion model) or 1/24.4 (Langevin model)14In liquid phase, the rotational correlation time τR1 by the conventional inversion-recovery method. Experiments were carried out at temperatures T = 353, 363, and 373 K and pressures 9 < p < 101 kPa. The translational diffusion of water molecules does not affect our measurements of longitudinal relaxation rates R1 (see Methods), although it might interfere with measurements of transverse relaxation rates R2. The rates R1 observed in neat water  and in a mixture of HDO and D2O  are reported in We measured longitudinal relaxation rates R1 shows a maximum at a pressure pmax where τJ = 1/(ωO − ωJ)  see .p can be estimated via the ideal gas law (see Methods) yielding τJ = RT/(pvσJ). Hence, it is possible to calculate the cross-section 2O:H2O collisions at pmax as p , all the other parameters being fixed to the values given in pmax = (17 ± 3) kPa provides a fair agreement between experimental relaxation rates (points) and predicted rates (lines)  . The spiC to values predicted by the Ivanov model (τC = 5/4τJ), extended diffusion model (τC = 1/4τJ) or Langevin model (τC = 1/24.4τJ)14C ≪ τJ), according to which significant contributions of p < 10 kPa.In a more refined analysis we included contributions eraction . We fixe2O mixtures, experimental relaxation rates R1 (sample 5) are reported in R1 in For HDO-D2O:H2O and HDO:D2O collisions are reported in Cross sections and correlation times for the transfer of the angular momentum in H2O-H2O and HDO-D2O collisions at pressures below 101 kPa and temperatures between 353 and 373 K. The ratio of cross-sections 2O and H2O:H2O collisions. This hypothesis is compatible with the fact that H2O and D2O have almost equal electric dipole moments2O and D2O152O is therefore not possible.Our analysis provides information about HThe collisional cross-sections calculated from our NMR data can be used to refine the anisotropic part of the intermolecular potentials for collisions in gas phase56572O (T > 373 K) into the cryostat, and the liquid HDO ‘bolus’, usually containing a hyperpolarized solute, is pushed by pressurized helium gas  through a polyethylene tube with a 1 mm inner diameter running through a “magnetic tunnel”averaged longitudinal relaxation rates are likely to be much shorter than those in liquid water. The shortening of T1 would lead to a rapid loss of hyperpolarization during the transfer between the polarizer and the NMR magnet. Note that the viscosity and surface tension of the transferred liquid are difficult to control, since it consists of an aqueous solution containing analytes, polarizing agents like TEMPOL and glass-forming agents such as glycerol.Our findings may be relevant for Dissolution Dynamic Nuclear Polarization (D-DNP)2O:H2O and HDO:D2O collisions in the gas phase and evaluated the cross-sections for the transfer of the angular momentum which can be used to refine the intermolecular potentials.To summarize, we reported NMR relaxation rates due to binary HOur experimental setup consisted of a pair of coaxial glass tubes .Tbp = 384 K). Its deuterium signal allows one to lock the static field and to shim its homogeneity. The inner tube contained water that was frozen and flame-sealed under vacuum (p = 1 kPa). Four tubes of 3.5 to 4 cm length, labeled as samples 1, 2, 3 and 4, were filled with ca 0.1, 0.2, 0.3 and 4.5 mg H2O, determined with a precision balance . A fifth tube (sample 5) was filled with 2 mg of 98% D2O and 2% H2O (v:v), hence containing ca. 2% HDO. The inner tube was completely immersed in the solvent contained in the outer tube (1H NMR spectra are shown in T = 363 K.The inner tube with 5 mm outer diameter was held in the center of a 10 mm tube by holders made of PTFE (Teflon). The outer tube contained about 2 mL of deuterated toluene-d8 (boiling point ter tube  in orderAll NMR experiments have been performed on a Bruker Avance-II 800 MHz spectrometer equipped with a 10 mm BBO probe.p and the number density p = psat). In that case the pressure psat can be calculated using Antoine’s equationTo determine the pressure T is the temperature and A, B and C are sample-specific constants. When expressing the pressure in bar and the temperature in K, we assumed2O and D2O, since their vapour pressures are similar within 1% over our range of temperaturesp can be estimated provided that the equation of state of the gas is known a priori. We have compared where 2H2Cl4, 0.2% v:v) to the toluene-d8 in the outer sample tube. We calibrated the integral of the C2H2Cl4 reference peak (near ~ 6 ppm) with respect to the number density of sample 4 (saturated vapour). The pressures in samples 1 to 3 are then determined by scaling the peak intensities of the vapour peak with respect to sample 4. The error on the pressures is assumed to be ±10%. The active volume of the 5 mm inner tube has been estimated from documentation by the manufacturer (Wilmad) to be 0.4 cm3.The quantity of water vapour in samples 1 to 3 has been determined by integration of the relevant signals in the NMR spectra. As a reference for integration we added 1,1,2,2-tetrachloroethane  we performed the following test. The inner tubes were only a few mm longer than the active region of the 1H coil of the 10 mm probe which is about 3 cm long. We measured R1 at the highest temperature T = 373 K (where the effects of diffusion are most pronounced) in two arrangements. First, we centered the inner tube with respect to the active region of the 1H coil. In this configuration, molecules can diffuse to and from the volumes above and below the active region. Secondly, we moved the inner tube up so that its bottom end was aligned with the lower end of the active region of the coil. In this manner, only molecules that cross the limit of the active region of the rf coil from above can influence the NMR signal. Any difference in R1 observed with these two configurations should be due to diffusion or convection. We found the R1 values to be identical within their errors, suggesting that contributions from diffusion can be neglected. Since we immersed the inner tube completely in a liquid with a controlled temperature, we assumed that there was no significant temperature gradient, so that convection due to differences in density should be negligible. Nevertheless, the experimental errors of the relaxation rates were doubled to take into account uncertainties stemming from diffusion and convection.Translational diffusion of water molecules in gas phase is very fast. Translational motion of water molecules between the active volume of the How to cite this article: Mammoli, D. et al. Collisional cross-section of water molecules in vapour studied by means of 1H relaxation in NMR. Sci. Rep.6, 38492; doi: 10.1038/srep38492 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.