The expression of protein TRPV1 and pTRPV1 was increased, and Ca2+ was increased in the visceral hypersensitive group. NGF, TrKA in NGF antagonist group, PI3K, AKT, NF‐κB in PI3K inhibitor group, PLC‐γ in PLC‐γ inhibitor group were all almost not expressed. The relative expression of NGF, TrKA, PI3K, AKT, PLC‐γ and NF‐κB in NGF antagonist group was lower than that in visceral hypersensitivity group and NGF activator group (P < .01). The relative expression of NGF, TrKA, PI3K and AKT mRNA in NGF antagonist group was lower than that in the normal model group (P < .01). There was no significant difference in the relative expression of PLC‐γ and NF‐κB mRNA (P > .05). The expression level of MAPK, ERK1 and ERK2 in visceral hypersensitivity group was higher than that in PI3K inhibitor group and PLC‐γ inhibitor group. The normal group Ca2+ curve was flat, and the NGF agonist group had the highest Ca2+ curve peak. Calcium concentration in visceral hypersensitivity group was higher than that in PI3K inhibitor group and that in PLC‐γ inhibitor group was higher than that in NGF antagonist group. The binding of TrkA receptor to NGF activates the MAPK/ERK pathway, the PI3K/Akt pathway and the PLC‐γ pathway, causing changes in the fluidity of intracellular and extracellular Ca2+, resulting in increased sensitivity of visceral tissues and organs.To investigate the efficacy of sacral nerve stimulation (SNS) on nerve growth factor (NGF) mediated visceral sensitivity in normal rat and visceral hypersensitivity model rats. 120 male newborn rats were randomly divided into 6 groups: group A was normal model group; group B ~ F were all sensitized with acetic acid enema and grouped again. Group c2 was given NGF antagonist, d2 group was given NGF agonist, e2 group was given PI3K inhibitor, and f2 group was given PLC‐γ inhibitor. After treatment, the expression of NGF, TrKA, PI3K, AKT, PLC‐γ, NF‐κB, TRPV1, pTRPV1 and intracellular Ca It was initially used for the treatment of urinary incontinence and retention. In 1995, it was used by Matzel for the minimally invasive treatment of faecal incontinence.Nerve growth factor (NGF) is one member of the neurotrophic factor family. It is widely distributed in the central nervous system, autonomic nervous system and intestinal nervous system. NGF has a stable synaptic nutritional role in regulating the transmission of synaptic signals. NGF mainly binds to two kinds of membrane receptors, usually with high‐affinity receptor tyrosine kinase A (TrkA), regulates downstream signalling pathways, promotes neurotransmitter release, synaptic receptor expression and changes neuroplasticity, and plays an important regulatory role in the survival, growth, differentiation and function of neurons.In this study, acetic acid enema‐induced chronic visceral hypersensitivity rats were used as animal models. The expression of NGF, TrkA receptor, PI3K/Akt and PLC in colon and DRG before and after SNS were detected by Western blot and RT‐PCR. After antagonizing NGF, the above contents were detected and the effect of SNS on visceral sensitivity was evaluated. The visceral sensitivity of rats was evaluated by blocking the downstream signal pathways separately. To explore whether SNS can improve visceral sensitivity by reducing NGF down‐regulation of TrkA receptor, and further study whether SNS can improve visceral sensitivity by affecting downstream signalling pathways  through NGF/TrkA receptor. This study will help us to further understand the signal transduction mechanism of visceral hypersensitivity and provide a theoretical basis for the clinical application of neuromodulation in the treatment of IBS.22.1One‐Twenty SPF‐grade male newborn rats were fed at quiet environment with room temperature (22 ± 2℃), constant humidity (40%‐50%) and 12/12 hours of circadian circulation. One week after adaptive feeding, 120 healthy and clean SD rats were randomly divided into 6 groups: group A, group B, group C, group D, group E and group F, 20 rats in each group.Group A: Normal model group.Group B: The visceral hypersensitivity model was established by acetic acid enema sensitization, and half of the model rats in group B were numbered b1/b2, respectively.Group C: On the basis of the model group, half of the rats in group C were numbered c1/c2. Group C was given NGF antagonists.Group D: On the basis of model group, half of the model rats in group D were numbered d1/d2, and group d2 was given NGF agonists.Group E: On the basis of model group, half of model rats in group E were numbered e1/e2, and PI3K inhibitors were given in group e2.Group F: On the basis of the model group, half of the rats in group F were numbered f1/f2, and the f2 group was given PLC‐γ inhibitor.2.2At the age of 10 days, the neonatal rats were fed with 0.5% acetic acid solution 0.2 mL through intestinal perfusion with insertion of anus for 2 cm, while the neonatal rats in group A were fed with 0.2 mL saline through intestinal perfusion. Electrode implantation was performed at the age of 7 weeks in rats, and the experiment began at the age of 8‐12 weeks.2+ in the cells of dorsal calcaneal ganglion and colon was detected by fluorescence spectrophotometer with fura‐2 indicator. The expressions of NGF, TrKA, PI3K, AKT, PLC‐γ, NF‐κ B, TRPV1 and pTRPV1 in dorsal root ganglion and colon tissues of animals in groups A, b2, c2, d2, E2 and F2 were detected by WB. Expressions of NGF, TrKA, PI3K, AKT, PLC‐γ, NF‐κ B, TRPV1 and pTRPV1 were analysed by real‐time fluorescence quantitative polymerase chain reaction (RT‐PCR). Co‐Immunoprecipitation (CO‐IP) was used to analyse the interaction between NGF and TrK A in dorsal root ganglion and colon tissues of animals in groups A, b2, C2 and d2, and fluorescence spectrophotometer was used to detect the intracellular Ca2+ content in dorsal heel ganglion and colon tissues of animals in groups A, b2, c2, d2, E2 and f2.The expression of TRPV1 and pTRPV1 in dorsal root ganglion and colon tissues of group A and group b1 were detected by Western blot (WB). The content of Ca2.3P < .05 was statistically significant.SPSS 18.0 statistical software was used to process and analyse the data, and the measurement data were described by mean ± standard deviation. Single factor analysis of variance was used for multi‐group comparison, and correlation analysis was used for correlation analysis. 33.1As shown in Figure 3.22+ in dorsal ganglion and colon tissue cells was detected by fluorescence spectrophotometer with fura‐2 indicator. The left figure in Figure 2+ content in dorsal calcaneal ganglion cells, and the right figure shows the intracellular Ca2+ content in colonic tissue cells. In both dorsal ganglion and colon tissues, the intracellular Ca2+ content in group b1  was higher than that in group A .The content of Ca3.3As shown in Figure 3.4−ΔΔCT, ΔCT was the target gene CT value‐internal reference (GAPDH) CT value, and ΔΔCT was the target sample ΔCT‐control sample ΔCT. The expression levels of NGF, TrKA, PI3K, AKT, PLC‐γ and NF‐κB in different groups were analysed by one‐way ANOVA. The results in Figure P < .01). The results of LSD showed that there was no significant difference in the relative expression of PLC‐γ and NF‐κ B between group A  and group c2 . There was no significant difference in the relative expression of PLC‐γ between group A  and group b2 . There was no significant difference in the relative expression of PLC‐γ between the other two groups (P = .08 > .05). The difference was statistically significant (P < .05).RT‐PCR was used to analyse the expression of the target gene. The relative expression of the target gene was analysed by CT. Normal or negative samples were usually used as control samples. The relative expression of NGF, TrKA, PI3K, AKT, PLC‐γ and NF‐κB was 23.5As shown in Figure 3.62+ in dorsal calcaneal ganglion and colon tissue cells was measured by fluorescence spectrophotometer with fura‐2 indicator. As shown in Figures 2+ in both dorsal calcaneal ganglion and colon tissue cells was flat in the normal group in Figure The content of Ca4Visceral hypersensitivity (VH) refers to the increased responsiveness and sensitivity of visceral tissues to various endogenous or exogenous chemical or physical stimuli. Increased visceral sensitivity is one of the main pathophysiological mechanisms of IBS‐D and is an important cause of abdominal pain, gastrointestinal motility disorders and clinical symptoms. Nonnoxious and harmful receptors are widely distributed in various organs of the body. The intestinal wall receptor can be directly contacted with the contents of the intestinal cavity. When the intestinal wall receptor is stimulated by noxious substances , the intestinal wall receptor transmits the stimulus signal along the afferent nerve to the dorsal root ganglion, which is regulated by the central visceral sensory region to produce visceral pain.2+), further activate protein kinase C (PKC) and play its catalytic role in protein phosphorylation.NGF is a kind of protein that can induce the growth of menstruation and is closely related to the development of nervous system.Previous studies have reported that PI3K/Akt and PLC signalling pathways play an important role in TNBS‐induced visceral hypersensitivity in rats, possibly through up‐regulating neuroreceptors such as NMDA receptors, promoting the release of SP, CGRP and other peptide transmitters, and altering neuroplasticity involved in the process of spinal sensitization to visceral hypersensitivity. The results of this study also confirm this point.2+ increased in visceral hypersensitivity group. NGF antagonist was given to visceral hypersensitive rats, and protein NGF and TrKA were hardly expressed. PI3K inhibitor was given, and protein PI3K, AKT and NF‐κ B were hardly expressed. The relative expressions of NGF, TrKA, PI3K, AKT, PLC‐γ and NF‐κ B in NGF antagonist group were lower than those in visceral hypersensitivity group and NGF activator group (P < .01). The expression levels of MAPK, ERK1 and ERK2 in visceral hypersensitivity group were higher than those in PI3K inhibitor group and PLC‐γ inhibitor group. The Ca2+ curve of normal group was flat, and the peak of Ca2+ curve of NGF agonist group was the highest. It has also been reported that PI3K/Akt and PLC signalling pathways play an important role in TNBS‐induced visceral hypersensitivity in rats. They may participate in visceral hypersensitivity by up‐regulating neuroreceptors such as NMDA receptors, promoting the release of SP and CGRP, and altering neuroplasticity. It is also found that MAPK/ERK signalling pathway does not participate in spinal cord hypersensitivity by up‐regulating the expression of NMDA receptors.In this study, we found that the expression of TRPV1 and pTRPV1 and the concentration of Ca2+ was changed. In addition, tissue damage can lead to electrolyte disturbance inside and outside the cell, which can also lead to the opening of transport channel TRPV1, the change of fluidity of intracellular and extracellular Ca2+ and the increase of the sensitivity of visceral tissues and organs.To sum up, the visceral hypersensitivity model was established by acetic acid enema sensitization. Intestinal tissues and intestinal nervous system were sensitized by external noxious stimuli. Intestinal nerve cells, intestinal epithelial cells and intestinal smooth muscle cells were activated to produce and release NGF. NGF combined with TrKA mainly caused to the opening of transport channel TRPV1 through PI3K/Akt and PLC signalling pathways, and the fluidity of intracellular and extracellular CaAll of the authors have no conflict of interest in this research.Each author has made an important scientific contribution to the study and has assisted with the drafting or revising of the manuscript.2+ fluidity and increasing visceral organ sensitivity.TrkA receptors bind to NGF by activating the MAPK/ERK pathway, PI3K/Akt pathway and PLC‐γ pathway, causing changes in intracellular and extracellular CaEthical approval was given by The First Affiliated Hospital of Nanjing Medical University.All of the authors have consented to publish this research.


We describe how the rates of T1 relaxation for the remaining 1H nuclei are increased and show how this leads to a concomitant increase in the level of 1H and 13C hyperpolarization that can ultimately be detected.By introducing a range of  In SABRE, parahydrogen (p‐H2) adds to a precatalyst, such as [IrCl(COD)(IMes)] imidazol‐2‐ylidene and COD = cis,cis‐cycloocta‐1,5‐diene), in the presence of a coordinating substrate (sub) to form an iridium hydride complex such as [Ir(H)2(IMes)(sub)3]Cl (2)parahydrogen derived hydride ligands is then transferred into the bound substrate through the J‐coupling network that exists in this complex.parahydrogen and the substrate, which are located in the bulk solution, are in reversible exchange with the corresponding ligands that are bound to the iridium complex. This results in a build‐up of hyperpolarized substrate in bulk solution and hence the signal gain grows with parahydrogen exposure time, although a limit is reached because relaxation acts to destroy the hyperpolarization that has been created through SABRE.1H nuclei but the wide array of heteronuclei that they contain.Hyperpolarization techniques overcome the inherent insensitivity of nuclear magnetic resonance (NMR) and magnetic resonance imaging by manipulating the spin distribution across Zeeman‐split energy levels away from equilibrium where a typical population difference used to produce a magnetic resonance (MR) signal can be less than 1 in 100,000. The employment of hyperpolarized states can therefore result in very substantial MR signal gains. This process can be achieved through a number of methods, the most common of which are dynamic nuclear polarization2H labelling of substrates such as pyridine,T1 relaxation times of the remaining 1H nuclei through inhibition of scalar relaxation processes.2H labelling of the IMes ligand of the iridium catalyst, the relaxation times of the bound substrate increase which further results in a dramatic improvement in the 1H hyperpolarization levels that can be achieved through SABRE. In fact, they exceed 50% polarization when 5 bar of parahydrogen is used in conjunction with a co‐ligand.2H labelling strategy by applying it to the related compounds pyridine, methyl isonicotinate, and isonicotinamide. We seek to ascertain its effect on the efficacy of the resulting 1H and 13C hyperpolarization levels that can be achieved through SABRE. For synthetic procedures and characterisation data please see It has recently been shown that the selective, partial 23) was one of the first compounds to be hyperpolarized by the SABRE method.2H labelling on the level of hyperpolarization. A selection of partially deuterated pyridines shown in Figure 3a, 3b, and 3c, in which 1H nuclei are retained at the ortho, meta, and para positions of the ring, respectively.Pyridine  is mixed with [IrCl(COD)IMes] (5 mM) and parahydrogen in ethanol‐d6 and shaken at 65 gauss for 10 s followed by immediate transport into the spectrometer for detection, the signal for Ha in Figure b and Hc are 1,120 and 1,258 times larger than those of the corresponding reference signals (at 9.4 T). Figure a polarization, created after just 10 s of exposure to 3 bar of parahydrogen. Furthermore, we note that if 20 s were needed to repeat the control measurement which uses thermally equilibrated polarization levels, it would take 1,333 days of data averaging to reproduce the hyperpolarized signal intensity. It is for this reason that hyperpolarization reflects a method that could transform clinical diagnosis as it may facilitate the facile detection of markers that probe underlying physiology.When d3‐pyridine (3a) was employed under identical conditions to those described for 3, the signal enhancement seen for the remaining ortho protons increases to 4,166 (6.7% polarization), shown in Table d3‐pyridine (3b), the remaining meta protons exhibit a smaller gain associated with an enhancement of just 541 (0.9% polarization). Finally, 2,3,5,6‐d4‐pyridine (3c) results in very little signal gain with its remaining proton showing a 163‐fold enhancement (0.51% polarization). These values are the average of three distinct measurements and are hence reproducible.When the agent 3,4,5‐3b and 3c can be rationalized as the result of the fact that their protons are magnetically isolated from those of the hydride ligands in the iridium catalyst. Hence, smaller J‐couplings are involved in the polarization transfer step and less effective SABRE transfer results.3a features two ortho1H nuclei that couple more strongly to the hydride ligands in the SABRE‐catalyst and they are therefore able to more readily receive magnetization. An analogous trend was found when methanol‐d4 was used as the solvent, and in this case, a polarization level of 5.8% could be achieved with 3a.The lower polarization levels seen for J‐coupling is somewhat offset by relaxation changes. At 400 MHz, in degassed ethanol‐d6, protio‐pyridine (3) was found to exhibit T1 relaxation times of between 18.2 and 26.7 s  in the presence of 1 for a 20::1 loading after SABRE at approximately 0.5 G. The resulting fully coupled single‐scan 13C hyperpolarized NMR spectrum is shown in Figure 13C counterpart, which is a 2,048 scan average. A significant increase in the 13C resonances' signal‐to‐noise ratios is clearly observed in the hyperpolarized spectrum. Furthermore, the ortho peak (148 ppm) is observed as an antiphase doublet of doublets, whereas the meta (124 ppm) and para (137 ppm) peaks are split into antiphase triplets of doublets. This is consistent with the creation of IzSz terms, where the antiphase component is associated with the small, indirect JHC coupling. The meta and para signals are associated with the detection of a 13CD signal for the meta and para sites, which accounts for the in‐phase 1:1:1 splitting.A series of hyperpolarized 1H decoupling a much weaker signal was observed  and isonicotinamide (5). A range of doubly deuterated isotopologues of these compounds were synthesized according to Scheme 1H spin systems, and we expected them to exhibit markedly different hyperpolarization characteristics. This reflects the fact that 4a and 5a possess pairs of ortho and meta1H nuclei that exhibit a mutual three‐bond coupling, in contrast, 4b and 5b contain two weakly coupled ortho and meta nuclei (5‐bond coupling), whereas 4c, 5c, 4d, and 5d contain pairs of equivalent ortho and meta nuclei, respectively.Following these observations, we turned our attention to the 4a and 5a) resulted in limited changes in their relaxation times relative to their 1H counterpart and somewhat similar polarization levels as a result of SABRE were observed  does indeed result in significantly improved relaxation times and SABRE polarization levels , respectively, to 11.0% and 11.1%, respectively. In this case, the extension of the T1 values determined for the meta proton in the presence of the SABRE catalyst greatly assists in increasing the observed polarization level of the proton at this position.For these ester and amide substrates, deuteration at the 2‐ and 3‐positions 4a and a resulteortho1H nuclei (4c and 5c) gave slightly improved levels of polarization and T1 values that compare to those of the parent, but still suffer from short relaxation times in the presence of the SABRE catalyst. This is consistent with the results outlined earlier for 3a. Compounds 4d and 5d, with only meta1H nuclei now give higher T1 values, but as expected, the improvement in achieved polarization level is minimal, in accordance with the weaker J‐coupling that connects them to hydride ligands in the catalyst.In general, compounds with only 1H nucleus at the ortho position result in efficient polarization transfer and that 1H nuclei that are isolated from each other, and the hydride ligands of the metal catalyst, lead to increased relaxation times under SABRE. These changes contribute cooperatively to produce a strong, long‐lived hyperpolarized signal.These trends confirm our earlier conclusions that substrates containing a 13C NMR experiments on the labelled isonicotinamides (5a–5d) showed that superior signal enhancements could be achieved with these substrates , four isotopologues of methyl isonicotinate (4), and four isotopologues of isonicotinamide (5). We find that harnessing hyperpolarization sites adjacent to the nitrogen centre of pyridine, which binds to the SABRE catalyst metal centre, is crucial for efficient polarization transfer through its stronger 4JHH coupling to the parahydrogen‐derived metal hydride ligands. Furthermore, we confirm that the presence of spin‐isolated hyperpolarization sites in these agents both increases signal lifetime through reduced relaxation and allows the detection of strongly hyperpolarized 1H responses. These changes also facilitate the detection of strong 13C NMR signals, most notably for the corresponding quaternary and CD positions. These signals typically appear with anti‐phase character due their origin in a heteronuclear longitudinal two spin order term (1H─13C) involving an indirect coupling. Agent 5b produced the strongest carbonyl signal as a consequence of a 3JHC coupling, which must lead to limited internal peak cancelation due to its small value. The 13CH signals of 5c are also dramatically stronger than those of 5a, 5b, and 5d in accordance with a predicted long relaxation time. The spin isolation of the C─13CONH2 groups in 5c and 5d though acts to reduce its detectability, although the signal for C─13CONH2 is strongly visible in 5a and 5b. Hence, we conclude that a 2H‐labelling strategy can be used to control not only 1H signal gains but also those of 13C. We expect that this strategy will enable improvement in polarization in molecules containing other heteronuclei, such as 15N, and work is ongoing to achieve this goal. Additionally, a detailed investigation into the mechanism of polarization transfer in molecules containing 2H nuclei would be of interest.An investigation has been made into the effect of selectively incorporating Data S1 Supporting InformationClick here for additional data file.


For such samples, the water contact angle (WCA) was shown to transition from a superhydrophobic (WCA ≈ 165°) to a superhydrophilic (WCA ≈ 0°) state within 10 min upon UV illumination and subsequent recovery to superhydrophobicity occurred after heat treatment. It was found that the changes in the trimethoxysilane upon UV illumination can explain the rapid decrease of the WCA from more than 165° to almost 0°. To further investigate the wettability transition, trimethoxysilane and Al2O3 nanoparticles  were mixed and spray-coated onto the glass substrates as the control samples. Then the unrecoverable change of trimethoxysilane under UV illumination can be confirmed. It was found that the presence of trimethoxysilane in the TiO2–trimethoxysilane coating served to speed up the super-wettability transition time from superhydrophobicity to superhydrophilicity, but also limited the number of wettability recycle times. With this understanding, the effect of the trimethoxysilane concentration on the number of recycle cycles was investigated.Controllable wettability is important for a wide range of applications, including intelligent switching, self-cleaning and oil/water separation. In this work, rapid switching and extreme wettability changes upon ultraviolet (UV) illumination were investigated. TiO Chagas et al. [2 modified with trimethoxypropyl saline. In this work, the wettability transition time from superhydrophobic (WCA ≈ 158°) to superhydrophilic (WCA ≈ 0°) occurred within 120 min of UV illumination.In order to be useful for many applications, the ability to rapidly switch the wettability from superhydrophobicity to superhydrophilicity is imperative. Several studies based on TiOtability . Wang ettability  preparedn et al.  modifieds et al.  fabricat2/fluoroalkyl end-capped vinyltrimethoxysilane sol–gel where the WCA changed from about 180° to 108° in under 50 min upon UV illumination and then further reduced to 20° after another 30 min. Thus the total transition time from superhydrophobic to superhydrophilic was about 80 min. Petroffe et al. [2 with 11-(4-(phenylazo)phenoxy)undecanoic acid (AzoC11) acid to fabricate a hybrid surface which could achieve a rapid change in wettability properties within 12 min, however the wettability range was only between hydrophobic (≈146°) and hydrophilic (≈21°). Qing et al. [2 nanoparticles with trimethoxysilane, which not only exhibited reversible wettability from 160° to 0° but also reduced the transition time to 60 min.Although large range wettability switching can be achieved in many ways, the transition from superhydrophobic to superhydrophilic always requires a longer time, ranging from a couple of days to a couple of hours –27. Somee et al.  modified2-based surfaces can achieve a faster change from superhydrophobic to superhydrophilic. However, the reason of the faster transition process has not explained. Due to the high energy of UV illumination and the photocatalysis effect of TiO2, the –CF2−, –CF3 groups of the modified trimethoxysilane might become photodegraded and the end of the trimethoxysilane would change into –OH groups without reversibility [2 nanoparticles and trimethoxysilane.By modifying trimethoxysilane, TiOsibility –32. It c2 nanoparticles and non-photosensitive Al2O3 nanoparticles as the control groups, respectively. The nanoparticles were modified with a short carbon chain trimethoxysilane, 1H,1H,2H,2H-perfluorooctyl(trimethoxy)silane (PFOS), in different concentrations. The wettability and the chemical bonding of the surfaces were tested upon UV illumination and heating treatments. The results are in agreement with the proposed assumption that the oxidization of the trimethoxysilane could decrease the super-wettability transition time, however the reversibility will be limited due to the irreversible oxidization process. The effect of trimethoxysilane concentration on the number of times the surface can be recycled was also investigated.In this study, to confirm this assumption, a series of experiments were carried out. Surfaces were coated with TiO2 nanoparticles were modified in ethanol solutions of PFOS, and the suspensions were sprayed on glass substrates to make the TiO2–PFOS samples. With the same method, Al2O3–PFOS samples were prepared as control groups. As shown in 2–PFOS and Al2O3–PFOS coated surfaces exhibited excellent superhydrophobicity, and the WCA was measured as about 165° and 171°, respectively. Based on the SEM images shown in The hydrophilic TiO2 nanoparticles were subjected to chemical bonding as Si–O–Ti with hydrolysing PFOS. The chemical bonding is verified by the Fourier transfer infrared (FTIR) spectra of TiO2 and TiO2–PFOS coated glass surfaces, as shown in −1 which further confirmed the dehydration reaction occurred between the hydrolytic PFOS and TiO2. Additionally, there were another three peaks at 1157, 1207, 1243 cm−1, which correspond to the stretching vibration of –CF2− and –CF3 groups [2− and –CF3 groups appeared in the FTIR spectra of Al2O3–PFOS but without the peak at 1065 cm−1, which indicates that there was probably no chemical bonding between Al2O3 and PFOS, suggesting the physical adhesion between Al2O3 nanoparticles and PFOS.The TiO3 groups . Similar2–PFOS coated surfaces and Al2O3–PFOS coated surfaces were directly illuminated under a UV lamp at a working distance of 10 mm, and the WCAs were recorded every 5 min. The TiO2–PFOS coated surface transitioned from superhydrophobicity to superhydrophilicity after only 10 min of UV exposure, while it took 90 min for Al2O3–PFOS surfaces to transition. Then the samples were heated at 150 °C for 90 min, the property of superhydrophobicity was recovered for the TiO2–PFOS coated surface, while the Al2O3–PFOS coated surface remained superhydrophilic. A transition time of 10 min from superhydrophobic to superhydrophilic is much faster than in previous works [2–PFOS surface, as the UV exposure time increases, the peak intensity at 1629 cm−1 and 3436 cm−1  increased, while both peaks reduced after the heating treatment. At the same time, four peaks at 1207 cm−1, 1243 cm−1, 2850 cm−1 and 2919 cm−1 (attributed to the bending vibration and stretching vibration of –CF2- and –CF3 groups) undergo no obvious changes either under UV illumination or high temperature exposure. The change in transmittance for selected transitions are listed in 2 is much larger than the change of –CF2- or –CF3 in PFOS. The change of –CF2- or –CF3 can be observed after long time UV illumination as shown in 2 and PFOS (Si–O–Ti) is without exception. The Al2O3–PFOS samples achieved the transition to super-wettability under UV illumination but required a much longer time of up to 90 min. The bands at 1210 cm−1 and 1240 cm−1 which represent the vibration of the –CF2- and –CF3 groups in PFOS was not recovered, while the –OH vibration in Al2O3 experienced no significant change.Both TiOus works –27. The 2 under UV illumination and heat treatment has been previously reported and was explained by the formation of Ti–O–H bonds under UV light, while heating of the surface results in the decrease in the concentration of Ti–O–H bonds [2, the photo-induced electron–hole pairs are generated on the TiO2 surfaces under UV illumination. The holes lead to the production of oxygen vacancies to enhance the adsorption of hydroxy groups, while the hydroxy groups are replaced by oxygen atoms that have a stronger bond on the defect sites during heating process. However, the change in TiO2 cannot fully explain the fast wettability transition of the TiO2–PFOS coated surface. Based on the data shown in 2- and –CF3 groups of PFOS were degraded and oxidized due to the high energy of UV illumination. The oxygen in the air stimulates a metastable state which has a strong oxidizing property. In addition, the end of the fluorocarbon chains were changed into –OH groups which could decrease the WCA faster with increasing UV illumination time. It can thus be inferred that the oxidization of PFOS is due to UV illumination and the photocatalytic effect of TiO2. Unlike the changes occurring in TiO2, unfortunately, this change in PFOS is not reversible. The mechanism for the wettability transition of the TiO2–PFOS coated surface under UV illumination and heating treatment is shown in 2- and –CF3 groups are replaced by the –OH groups on PFOS. PFOS has a short carbon chain which can enhance the change of the –CF2- and –CF3 groups and results in the much faster conversion time from superhydrophobicity to superhydrophilicity compared with the previous works [2- and –CF3 groups by –OH groups on PFOS was also confirmed by the wettability transition of the Al2O3–PFOS coated surfaces.The wettability transition of TiO–H bonds –34. As rus works –36. The 2 modified by different concentrations of PFOS were sprayed onto the glass samples. All the coated surfaces were initially superhydrophobic. Then the coated samples were placed under UV illumination until they transitioned to superhydrophilic, and were thereafter heated in an air-dry oven until they recovered their superhydrophobic property – this cycle was repeated until the surfaces could no longer recover the superhydrophobic property. For each concentration of PFOS, five identical samples were prepared for statistical reliability. The experimental results are shown in 2-, –CF3 and can initially increase the maximum number of conversion cycles. However, an excess of PFOS results in an increase in the time to achieve superhydrophilicity which enhances the amount of –CF2-, –CF3 changed in one cycle, and subsequently decreases the number of possible cycles. As a result, to obtain a faster wettability transition time as well as a larger number of conversion cycles, an appropriate amount of PFOS and TiO2 is needed. It should be noted that with the same concentration of PFOS, the total UV illumination time for the surface to transition to unrecovered superhydrophilicity for TiO2–PFOS and Al2O3–PFOS is 30 min and 90 min, respectively. This indicates that the oxidization time of PFOS is increased by the photocatalytic effect of TiO2.To further study the wettability switching of the surfaces, four samples comprised of a uniform weight of TiO2- and –CF3 groups, which is also in agreement with the proposed mechanism in For the sample with 1.5 g PFOS, the CA of the six cycles and the images of the conversion processes in the first and the last cycles are shown in 2 nanoparticles modified with PFOS (which has relatively short carbon chains) were applied on glass substrates to achieve fast, switchable super-wettability, demonstrated by a WCA that could be recovered from ≈165° to almost 0°. The experimental results show that the PFOS could reduce the conversion time from nearly 1 h to about 10 min, and the superhydrophobicity of the surfaces could be recovered after heating for 90 min at 120 °C. However, it was shown that certain concentrations of PFOS limit the number of recovery cycles, whereby the optimal concentration of PFOS was identified to achieve about 5 to 10 cycles. The unrecoverable change of the –CF2- and –CF3 groups on PFOS and the recoverable change of –OH groups on TiO2 were recorded by FTIR. These effects can explain both the shorter transition time and the limitation that  the change of the –CF2- and –CF3 groups leads to a quicker conversion time from superhydrophobicity to superhydrophilicity. However, this is only true when most of the –CF2- and –CF3 groups are changed unrecoverably, and the whole surface cannot not transition back to a superhydrophobic state.TiO2, rutile) with a diameter of ≈25 nm and aluminium dioxide (Al2O3) with a diameter of ≈30 nm were acquired from Shanghai Aladdin Bio-Chem Technology Corporation . 1H,1H,2H,2H-Perfluorooctyl(trimethoxy)silane (PFOS) was obtained from Shanghai Macklin Biochemical Corporation . Other chemicals include 99.9% ethanol used as the solvent and deionized water was used in the CA measurements.Titanium dioxide . When the surfaces were dried, the superhydrophobicity is revealed. Al2O3 was modified by the same method as TiO2.Rutile phase TiO2 and the wavelength was 254 nm. The change of the WCA under UV light illumination was measured at short intervals. For the heating process, which was employed to recover the superhydrophobicity, the surface was heated in the air-dry oven at 150 °C for 90 min after UV illumination. The switching time from superhydrophilic to superhydrophobic was found to be reduced with increasing heating temperature.A UV lamp (30 W) obtained from Cnlight Optical-Electrical Technology Corporation  was used to generate the UV illumination. The ultraviolet power density was set at 94 µW/cmThe microstructure of the coating surfaces was studied using scanning electron microscopy , and the surface roughness was measured with a laser confocal microscope . The chemical modification and the end group changes on the surfaces was studied by Fourier transform infrared spectroscopy . The contact angles were measured at room temperature with an optical contact angle meter , where the static CAs of the droplets (6 µL) placed onto the surfaces were measured five times at different locations.


Conversely, in the presence of ATP and in the absence of a pmf, the enzyme works as an ATP-driven proton pump. Here, we investigate how high concentrations of ATP affect the enzymatic activity of the F1F0 ATP synthase under high pmf conditions, which is the typical situation in mitochondria or growing bacteria. Using the ATP analogue adenosine 5′-O-(1-thiotriphosphate) (ATPαS), we have developed a modified luminescence-based assay to measure ATP synthesis in the presence of millimolar ATP concentrations, replacing an assay using radioactive nucleotides. In inverted membrane vesicles of E. coli, we found that under saturating pmf conditions, ATP synthesis was reduced to ~10% at 5 mM ATPαS. This reduction was reversed by ADP, but not Pi, indicating that the ATP/ADP ratio controls the ATP synthesis rate. Our data suggests that the ATP/ADP ratio ~30 in growing E. coli limits the ATP synthesis rate to ~20% of the maximal rate possible at the applied pmf and that the rate reduction occurs via product inhibition rather than an increased ATP hydrolysis rate.Synthesis of ATP by the F The pmf, composed of a proton gradient (ΔpH) and a membrane potential (Δψ), energizes the rotary mechanism of the F1F0 ATP synthase that catalyzes the synthesis of ATP from its substrates ADP and Pi . The F1F0 ATP synthase, which in most organisms can also work as an ATP driven proton pump, is composed of the membrane embedded F0 part and the soluble F1 part and can be divided into a stator and a rotor part. During ATP synthesis, when the pmf is high, protons are transported through the interface of subunit a and the c-ring converting the pmf into mechanical rotational energy. The c-ring is connected with subunits ε and γ forming the rotor, which transmits the rotary energy to the catalytic sites in the α3β3 headgroup. In the absence of a pmf, the enzyme can hydrolyze cellular ATP to establish an electrochemical gradient (the enzyme rotates in the opposite direction). This hydrolytic function of the F1F0 ATP synthase is vital for some bacteria under anaerobic conditions to maintain the membrane potential, when substrate level phosphorylation is the only source of ATP.Life is energetically expensive and mostly paid in adenosine triphosphate (ATP). In all aerobic organisms, reducing equivalents from the oxidative breakdown of nutrients are converted to ATP in a process termed oxidative phosphorylation. Located in the cytoplasmic membrane of bacteria or the inner membrane of mitochondria, the members of the respiratory chain create and maintain a proton motive force  The most popular and sensitive technique is based on the luciferase/luciferin couple, which in the presence of oxygen and ATP emits light that can be detected by a luminometer . The technique is very sensitive and is applied to determine the total ATP content from all types of samples. However, it cannot be used to measure the synthesis of newly formed ATP in the presence of physiological concentrations of background ATP (mM) which is ~1000-fold too high. Consequently, very little to no ATP is present in kinetic ATP synthesis measurements that are followed by this method. (II) The problem is circumvented by the second method, where radioactively labeled 32PO42− or 33PO42− is added to the solution. Newly synthesized ATP will incorporate radiolabeled phosphate, while the already present ATP is not radioactive. After separation of excess labeled phosphate by extraction, the newly formed ATP can be quantified via autoradiography or scintillation counting. The drawbacks are the use of radioactive substances and the laborious workup that prohibits continuous measurements. (III) The third method is based on the conversion of produced ATP by coupled enzymatic reactions that can be detected by spectroscopy, e.g. addition of D-glucose in the presence of hexokinase and subsequent dephosphorylation by glucose-6-phosphate dehydrogenase in the presence of NAD(P) that is reduced to NAD(P)H and can be followed spectroscopically. Similar to luciferase, this method cannot discriminate between present or freshly synthesized ATP and is less sensitive than the other methods. (IV) A further and more indirect technique originally developed by Chance and colleagues22 to quantify ATP synthesis is to measure the alkalization in the surrounding buffer solution during ATP synthesis. During ATP formation from ADP and Pi, a water molecule is formed that requires uptake of a proton from solution that can be measured either by a pH electrode or a pH sensitive dye such as phenol red. This approach is insensitive towards high concentrations of nucleotides and has successfully been used. However, it comes with limitations that have to be carefully addressed, such as low buffering capacity, limited pH range, and contribution of other proton dependent processes32.In contrast to ATP hydrolysis that can be investigated in detergent solution or even with the F1F0 ATP synthase, but is not detected by the luciferin/luciferase system. With the developed method, we were able to measure and compare the ATP synthesis in inverted E. coli membrane vesicles as a function of ADP, ATPαS, and inorganic phosphate (Pi) concentrations. We observe that physiological concentrations of ATP (5 mM), under the form of ATPαS, reduce the ATP synthesis rate to ~10%, and that this rate can be restored using elevated ADP concentrations, but not Pi, suggesting that the ATP synthesis is controlled by the ATP/ADP ratio and not by the ATP/(ADP∙Pi) ratio. The applicability of the technique to more isolated systems was confirmed in ATP synthesis experiments in liposomes containing purified bo3 oxidase and ATP synthase from E. coli.In the present work, we describe a modification of the luciferase/luciferin technique that allows rapid continous measurement of the ATP synthesis rate with a high time resolution at physiological (millimolar range) concentrations of ATP. The principle is based on the properties of a modified ATP, adenosine-5′-O-(1-thiotriphosphate), or ATPαS, which is a hydrolytic substrate for the Fin vivo. The luciferase first catalyzes the binding of D-luciferin to the α-phosphate of ATP-Mg2+, forming PPi-Mg2+ and luciferyl-AMP, which is subsequently oxidized by oxygen to form AMP, CO2 and oxyluciferin in an excited state that emits a photon while returning to the ground state . ATPases like the F1F0 ATP synthase, however, cleave ATP between the β- and the γ-phosphate, leaving ADP and Pi behind. We therefore reasoned that the use of an α-phosphate modified ATP prohibits the reaction with the luciferase but allows the reaction with ATPases. Indeed, commercially available ATPαS, in which the double-bonded oxygen on the α-phosphate is substituted for a sulfur, has been shown not to be a substrate for the luciferase in the deoxy form35, but is hydrolyzed by ATPases including the chloroplast F1F0 ATP synthase36 and the F1 ATPase from Bacillus PS337.The widely used reaction of D-luciferin with ATP, catalyzed by the luciferase from firefly, suffers from the drawback that it cannot discriminate between already present and freshly synthesized ATP. In addition, the high sensitivity of the assay impairs the use of ATP concentrations higher than a few micromolar, which is ~1000-fold less than what is found −12 M up to 5 × 10−3 M. The luminescence produced by the reaction of the luciferase reagent with ATP spanned from ~10° to ~108 arbitrary units (a.u.) between 1 pM and 5 mM whereas ATPαS yielded <104 a.u. at 5 mM  or to contaminating ATP in the ATPαS preparation. We also found that ATPαS concentrations above 100 µM decreased the overall luciferase signal (<50% at 1 mM), probably due to competition of ATPαS with ATP for the luciferase binding site. Experimentally, this effect is addressed by adding a known concentration of ATP (2.5 µM in our experiments) to relate the arbitrary units of the luminescent signal to the concentration of ATP for the quantification of ATP production. Figure We tested this hypothesis experimentally by comparing the reaction of ATP and ATPαS with the firefly luciferase/luciferin supplied as a mixture in a commercial kit  that is used by many to detect ATP synthesis. A typical amount of the assay was mixed with buffer and varying amounts of normal ATP or ATPαS were added to reach a final nucleotide concentration ranging from 10 mM Fig. , represeE. coli F1F0 ATP synthase in detergent solution  was 84% of that of ATP at the maximum tested substrate concentration . We also compared the ability of ATP and ATPαS to elicit transmembrane proton pumping, i.e. establishment of a pmf, with ACMA quenching in inverted membrane vesicles (IMV) of E. coli cells . Proton pumping was accelerated ~2.5-fold in the absence of a membrane potential  of E. coli, produced by a French Press39. In contrast to mitochondria, E. coli membranes do not contain nucleotide and phosphate carriers that control the availability and distribution of their substrates in the mitochondrial matrix. In IMVs, the orientation of all proteins is homogenously inverted40, exposing the F1 part of the F1F0 ATP synthase to the buffer solution, allowing to readily manipulate the relevant substrates ATP, ADP and Pi. For prospective studies investigating mutant variants, we used an E. coli F1F0 ATP synthase knockout strain (DK8), transformed with the plasmid pBWU13, constitutively expressing F1F0 ATP synthase. This combination has been shown to yield F1F0 ATP synthase overexpression when grown in minimal media using glycerol as carbon source41. If grown in rich media, however, F1F0 ATP synthase production levels were comparable to a strain BL21 with chromosomally encoded F1F0 ATP synthase, as judged by Western blot  and ubiquinone Q1 as synthetic electron donor and acceptor, respectively, to energize the quinol oxidase bo3 as the primary proton pump to establish a maximal pmf  to obtain maximal ATP synthesis was titrated , that was started by the addition of 80 µM ubiquinone Q1. ATP synthesis was immediately observed as a linear increase of the luminescence signal and was followed for at least 30 s. Addition of 400 nM potassium cyanide, a potent inhibitor of the bo3 oxidase, rapidly blocks ATP generation. Similarly, <10% ATP synthesis was observed in the presence of the proton ionophore CCCP (10 µM). Using 2 mM DTT and 80 µM ubiquinone Q1 as electron donor, the ATP synthesis rate was about 1.5 to 2-fold higher compared to energization with 500 µM NADH, both in the DK8/pBWU13 and BL21 strains  and Pi (100 mM) concentrations, the ATP synthesis rate declined to ~70%.In a first set of experiments, we characterized our experimental system by varying concentrations of inorganic phosphate Fig.  and ADP pmf. Keeping ADP (50 µM) and phosphate (10 mM) constant, we varied the ATPαS concentration between 1 µM and 5 mM, resulting in a distinct decrease in the synthesis rate. As depicted in Fig. Next, we tested the influence of increasing concentrations of ATPαS on the rate of ATP synthesis in the presence of a constantly high i]) influences the ATP synthesis rate. At chemical equilibrium, where no net ATP synthesis takes place the mass-action ratio is equal to the equilibrium constant K44. In equilibrium thermodynamics, the Gibbs free energy change is defined by the equation−1 at 50 µM ATPαS and 104 M−1 at 5 mM ATPαS) directly affect ATP synthesis rates. It was thus interesting to observe whether the effect caused by increased ATPαS concentrations could be reverted by increasing ADP or phosphate simultaneously, thus leaving Γ’ constant (Γ’ = 100 M−1). First, we tested this by increasing both ADP and ATPαS concentrations in parallel at a fixed Pi concentration (10 mM) to keep Γ’ = 100 M−1 and ATPαS/ADP = 1 constant , and slowly decreased to 60% (116 ± 18 nmol ATP min−1 mg−1) at 5 mM ATPαS and ADP. Considering the decrease of activity by 5 mM ADP itself , to keep Γ’ = 100 M−1 and ATP/Pi = 0.005 constant. The data from Fig. −1 mg−1 at 50 μM ATPαS (ATP/ADP = 1) to 37 ± 3 nmol ATP min−1 mg−1 (20 ± 3% of maximum observed rate) at 1 mM ATPαS (ATP/ADP = 20). This pronounced decrease is probably due to the inhibitory effect observed at high phosphate concentration . Proton pumping was initiated with DTT/Q1 and ATP synthesis was followed as described for the inverted membrane vesicle. First, ATP synthesis rates in the presence of 50 µM ADP, and either 10 µM or 2 mM ATPαS were measured. As depicted in Supplementary Fig. 1F0 ATP synthase.We have repeated the crucial experiments also with proteoliposomes containing F4 times lower than ATP) is due either to a very slow reaction of ATPαS with luciferin or to slight ATP contamination in the ATPαS preparation. ATPαS binding to luciferase/luciferin as a competitive inhibitor is likely, as the overall signal for a constant amount of ATP is decreasing with increasing concentrations of ATPαS. Technically, this effect was compensated by the addition of a precisely known, suitable amount of ATP for every measurement to quantify the luminescence signal. In contrast to its reaction with luciferin/luciferase, ATPαS behaved similarly to ATP when used to drive ATP hydrolysis with the E. coli F1F0 ATP synthase. As with the F1F0 enzyme in chloroplasts and the isolated F1 part of the ATP synthase of Bacillus PS3, ATP hydrolysis and proton pumping kinetics with the E. coli enzyme were comparable. In the reaction with ATPαS, Vmax was ~75% of the one found for ATP and both had a similar Km’ of ~200–300 µM in the absence of ADP. Earlier, Löbau et al. reported an apparent Km(MgATP) = 140 µM for E. coli F1F0 ATP synthase45. Data for ATPαS with intact F1F0 ATP synthase is missing, but in elegant single-molecule experiments following the rotation of the gamma subunit, Noji and coworkers have found an apparent Km (91 µM) with the purified F1 ATPase from Bacillus PS3 lacking the regulatory epsilon subunit37. In comparison to published values, the Km values reported here were ~2-fold higher, which is likely due to different assay conditions  and different enzyme preparations from different organisms. However, given the similar behavior within our preparation, it was concluded that ATPαS can be used to replace background ATP in ATP synthesis experiments. In the past, ATP synthesis experiments in the presence of high ATP were performed following the incorporation of radioactive 32PO42- or 33PO42- into newly formed ATP.In order to allow continuous detection of ATP synthesis by a luciferase/luciferin assay in the presence of high ATP concentrations, we have used a modified ATP analogue that is not detected by the luciferase system but is a suitable substrate for ATP hydrolysis with similar kinetic properties as ATP. We show that the sulfur-substituted alpha phosphate indeed barely reacts with luciferin. The observed signal increase upon rising ATPαS concentration (~10−5). However, the presence of respiratory proton pumps (e.g. bo3 oxidase in E. coli) generating and maintaining a pmf are able to shift the net reaction towards ATP synthesis. Typical in vitro ATP synthesis studies nevertheless employ conditions where no ATP is present due to the incompatibility with the luciferase assay, while ADP is used in excess to maximize the observed rates. In this report, we describe the use of ATPαS to overcome this limitation and have investigated the effect of cellular concentrations of ATPαS  on the enzymatic reaction of the E. coli F1F0 ATP synthase directly. Similar measurements have been performed in intact mitochondria, but the interpretation of results is complicated by the presence of pmf driven phosphate and nucleotide carriers from the SLC25 family that transport these substrates from the bulk to the site of ATP synthesis in the mitochondrial matrix. Consequently, the different substrates added to the outside affect the F1F0 ATP synthase only indirectly. By contrast, no such transporters are present in E. coli and the complete inversion of protein orientation in IMVs allows direct access of the experimentally added substrates ATP, ADP and Pi to the catalytic sites in the F1 part of the ATP synthase.The physiological nucleotide concentration ratio (ATP/ADP ~20–1000) strongly favors ATP hydrolysis  provokes a sharp decrease of ATP synthesis over an ATP/ADP ratio of 1, with 50% activity at ratio of ~20 (1 mM ATPαS) and <10% activity at a ratio of 100 (5 mM ATPαS), which is in good agreement with the data from mitochondria. As the pmf in our experiments is unchanged (no notable effect of increasing nucleotide concentrations on respiratory activity were found), the data indicates that the observed effect is either due to product inhibition by the presence of ATPαS or by an increased rate of ATP hydrolysis. Since our assay directly detects newly formed ATP with a very high time resolution and an immediate decrease in ATP synthesis rate is observed, we can confirm that a direct competition of ATPαS and ADP indeed takes place. We cannot rule out an increased ATP hydrolysis rate, but the effective ATPαS concentrations (>1 mM) are well above the apparent Km of ATP for ATP hydrolysis, which however could be increased in the presence of a pmf. In similar experiments, where ADP was raised in parallel to keep constant the ATPαS/ADP ratio and mass-action ratio (Γ’ = 100 M−1), the inhibitory effect of ATPαS was only mildly observed, suggesting that the competition of ADP and ATPαS indeed control the rate of ATP synthesis. A more detailed interpretation of these results is complicated by the presence of three catalytic sites that have different affinities for ATP and ADP depending on their conformation state 52. Finally, by varying the ATP and Pi concentrations in parallel, resulting in the same Γ’ = 100 M−1, but increasing the ATP/ADP ratio, we found that a variation of the phosphate concentration could not prevent the inhibitory effect, indicating that ATP synthesis is not controlled by the phosphorylation potential variable [ATP]/([ADP]∙[Pi]), but rather by the ATP/ADP ratio. The inhibitory effect of ATPαS at higher phosphate concentrations was even more pronounced , which can be attributed to a slight inhibitory effect of Pi concentrations >100 mM.With the present study, we can add information to the situation in E. coli, the ATP and ADP concentrations have been found to be ~3.5 mM and 0.12 mM, respectively, yielding an ATP/ADP ratio of ~3053. From our measurements, we can thus assume that despite saturating pmf, the E. coli synthesizes ATP with a rate of only ~20% of vmax. Etzold et al. determined a maximal turnover rate of 270 ATP s−1 per ATP synthase in E. coli at 37 °C54. However, in their measurements, 95% of the ATP synthases were blocked to maximize the available driving force per F1F0 ATP synthase. Furthermore, their experiments were performed in the presence of 1 mM ADP and the total absence of ATP (ATP/ADP = 0). It can thus be estimated that the ATP synthesis rates is only 5 to 10 ATP s−1 per F1F0 ATP synthase in growing E. coli. The observed necessary concentrations of ADP >1 mM to restore maximal ATP synthesis are not physiologically relevant. However, the nearly universal ATP hydrolysis inhibition mechanism by Mg-ADP59 could be responsible for the apparent absence of ATP hydrolysis in our experiments. We have thus measured the inhibitory effect of ADP on proton pumping on the same vesicles as used for ATP synthesis. The data from Fig. i of ADP increases from ~100 µM to ~400 µM in the presence of 50 µM ATP or 2.5 mM ATP, respectively. An even higher Ki is expected during ATP synthesis under high pmf, that has been shown to counteract ADP inhibition of ATP hydrolysis57, and hence being considerably higher compared to the ADP concentrations found in growing cells (~120 µM)53. ADP concentrations >400 µM, however will be easily reached when the pmf is low and ATP is converted to ADP, and the mechanism thus serves as effective protection against cellular depletion of ATP. Finally, our results seem to be independent of the reported inhibition of ATP hydrolysis by the ε subunit, as for the E. coli enzyme it has been shown to be absent in the presence of a pmf and independent of the ATP concentration14. In the future, it will be interesting to investigate, if ATP synthesis in other organisms than E. coli is similarly dependent on the ATP concentration.In respiring in vitro under physiological nucleotide concentrations and high pmf, using ATPαS as a replacement for ATP. The method allows to selectively measure newly produced ATP and is unaffected by the presence of high concentrations of ATPαS. The luciferase setup allows rapid and continuous measurements, avoiding the necessity of taking samples at distinct time points, thus considerably increasing time resolution in measurements.In this work, we have introduced a novel, alternative, non-radioactive method to measure ATP synthesis E. coli and proteoliposomes containing F1F0 ATP synthase and bo3 oxidase and find that the ATP/ADP ratio, but not ATP/(ADP∙Pi), controls the rate of ATP synthesis. The effect is based on a direct competition between of ATPαS and ADP for the catalytic binding sites. From this, we can conclude that the physiological ATP concentration prevents a maximal ATP synthesis rate despite saturating pmf conditions. Finally, our results suggest that Mg-ADP inhibition of ATP hydrolysis is not relevant to maximize ATP synthesis rates in E. coli, but is an effective tool to prevent total loss of cellular ATP during energy starvation.We have applied this method to inverted membrane vesicles of Chemicals and kits were purchased from Sigma-Aldrich and Thermo Scientific if not otherwise stated.E. coli strain DK8, lacking the unc operon coding for its ATP synthase, was transformed with a pBWU13 plasmid and grown at 37 °C in LB-Lennox medium containing 50 μg/ml ampicillin.2, and 10% glycerol), supplemented with a tiny spatula tip of Pefabloc serine protease inhibitor, phenylmethanesulfonyl fluoride and DNAse I, and broken with a French pressure cell press or a Microfluidizer. Unbroken cells were collected by centrifugation at 8000 g for 10 min. The supernatant containing the membrane vesicles was centrifuged at high speed (200′000 g) for 1 h, resuspended in 5 ml HMG supplemented with 0.5% cholate per gram of initial cells, to remove proteins loosely associated with the membrane, and centrifuged again at high speed. The membrane vesicles were resuspended in HMG and centrifuged a third time at high speed to remove traces of cholate. They were then resuspended in HMG to a concentration of 10 mg/ml total proteins (quantified with a bicinchoninic acid assay), aliquoted and flash-frozen with liquid nitrogen for storage at −80 °C.The following procedure was performed at 4 °C. Bacterial cells were suspended in 10 ml/g HMG buffer . The fluorescence of this membrane permeable dye quenches when a proton gradient forms across a lipid compartment60.With this method we indirectly measure the hydrolytic activity of the F2, 10 mM KNO3), 150 μg/ml inverted bacterial membrane vesicles and 2 μM ACMA in a final volume of 1 ml. The fluorescence baseline, taken as 100% fluorescence to determine the percentage of quenching, was measured and hydrolysis was started by the addition of 1–500 μM ATP or ATPαS. The pH gradient established by the ATP synthase quenched the ACMA dye proportionally to the hydrolytic activity and was eventually dissipated by the addition of 20 mM NH4Cl.The fluorescence of the dye 9-Amino-6-Chloro-2-Methoxyacridine (ACMA)  was monitored with a Cary Eclipse Fluorescence Spectrophotometer from Agilent Technologies. A cuvette with stirring magnet was filled with HMK buffer .Phenol red is a pH-sensitive dye which can be used to detect the dissociation of protons in the ATP hydrolysis reaction2, 0.1 mM EDTA, 10% glycerol, 0.02% triton X-100) was mixed with 25 μM phenol red and desired concentrations of ADP, ATP and ATPαS, to a final volume of 1 ml. The pH was adjusted to 8 with KOH and the baseline absorbance was recorded at 557 nm with a Cary 60 UV-Vis spectrophotometer. 20 nmol HCl were used for standardization and 5 μg E. coli purified F1F0 ATP synthase were added to start the reaction.Phenol red buffer  of an ATP Bioluminescence Assay Kit CLS II from Roche Applied Science was added to 50 mM Hepes-NaOH pH 7.5, 10 mM total phosphate and 3 mM MgCl2, and the baseline was measured. 1 μM to 5 mM ATP or ATPαS was then added and the luminescence measured.To compare the reactivity of ATP 2, 200 μg/ml luciferase reagent, 40 μg/ml inverted bacterial membrane vesicles, 2 mM (saturating) DTT and variable concentrations of ATPαS, ADP  and total phosphate . The luminescence was monitored with a Promega GloMax luminometer in steps of 30 s, allowing additions of relevant reagents to the tube when required.The assay was performed in clear 1.5 ml Eppendorf tubes in a final volume of 100 μl. 50 mM Hepes-NaOH pH 7.5 were used as buffer and the assay mixture contained 10 mM MgCl1, an ethanol soluble analogue of ubiquinone that can be reduced by DTT, was used to energize the membrane by bo3 oxidase proton pumping and start the ATP synthesis (3 × 30 s). ATP synthesis could be interrupted by the addition of 400 nM potassium cyanide or 10 μM CCCP (2 × 30 s).The baseline was first measured (30 s), before 250 pmol ATP were added as a calibration standard (30 s). Ubiquinone QSupplementary Information


Bluetongue is an arthropod‐borne viral disease of ruminants caused by bluetongue virus (BTV). In China, BTV is relatively common in Yunnan Province with the exception of northern regions around Shangri‐La, where the average altitude is approximately 3,450 metres. Recently, the seroprevalence of BTV has been measured in yaks in Shangri‐La; therefore, this study investigated BTV infections in this area. The serological investigation in five villages in Shangri‐La showed that there were sporadic BTV infections in yaks (20 of 507 positive) during 2014 to 2017, while the seroprevalence of BTV at three goat farms in a nearby river valley was 35%–65% in 2017. Subsequently, 20 sentinel goats were kept on two separate farms in the river valley and monitored for seroconversion between May and September of 2017. Five of the sentinel animals were tested positive for antibodies to BTV by C‐ELISA during the study period, and 13 BTV isolates were isolated from ten sentinel animals. All isolates were identified as the same serotype, and the complete nucleotide sequence of one was determined. The genomic sequences showed that the isolated BTV strain belonged to serotype 21 and had approximately 99.8%–100% homology with three Indonesian BTV‐21 strains  between their coding sequences (CDSs) except for Seg4 (99.5%). Besides, our data suggested that this BTV‐21 strain might have also infected some local yaks and sheep. It is the causative agent of bluetongue disease, which is an infectious, non‐contiguous and arthropod‐borne viral disease of ruminants  is the prototype of the genus nts Roy, . BTV wasnts Roy, . Some spThe BTV genome is composed of 10 segmented dsRNAs, which are named Seg1 to Seg10. The genomic RNAs encode seven capsid proteins (VP1‐VP7) and five non‐structural proteins (NS1‐NS4 and NS3a). The virion contains the 10 segmented dsRNAs and few non‐structural proteins in the core and is coated by two layers of capsids Roy, . VP2 andYunnan Province is located in a tropical and subtropical region and has diverse climates. It is the main region of BTV prevalence in China, and up to 13 serotypes of BTV have been identified in Yunnan  during 2014 to 2017 in 5 villages, and serum samples were collected from goats in 2017 from three different farms in the village of Nixi, which is near the other villages.+ blood from all sentinel animals were collected every 10 days and used for tests and virus isolation.After that, 20 local goats less than 1 year old that were identified as BTV negative by C‐ELISA were selected as sentinel animals and assigned to two surveillance sites in Nixi. The animals were kept among other goats between May and September of 2017. Serum samples and EDTA+ blood was collected from yaks and sheep in the villages of Xiaozhongdian and Nixi in 2017, and a series of BTV strains isolated in China previously and recently were also used for BTV Seg1 fragments alignment.In addition, for viral nucleic acid research, EDTA2.22.2.1sample/ODnegative. Samples with PI ≥ 50% were considered positive.The C‐ELISA method was used to test the anti‐VP7 (BTV) antibodies of animal serum samples, as described previously . Briefly, the lysates of BTV‐infected BHK‐21 cells were used to coat 96‐well microplates, and serogroup‐reactive anti‐VP7 mAb (8A3B6)  of the newly isolated virus was added at a volume of 50 μl per well and mixed with the diluted serum samples. After that, 1 × 104 BHK‐21 cells were added to each well in a volume of 100 μl, and after incubation for 4–6 days, the CPE was observed under an inverted microscope. The wells were scored for the degree of CPE observed. Titres were expressed as the final dilution of serum present in the serum/virus mixture when 50% of the duplicate wells were protected according to Reed–Muench methods  with a MagMAX‐96 Viral RNA Isolation Kit . A pair of universal primers, Bs1p3f (5‐TCATCGAATT TCGGGCTAA) and Bs1p3r (5‐CCCACATCTT TACAAACCAC), were used to amplify a 429 bp fragment of BTV Seg1. RT‐PCR for Seg1 was performed by a GeneAmp 9700 PCR system (Thermo) and a PS One‐Step RT‐PCR Kit ver.2 (Takara) using primers Bs1p3f and Bs1p3r according to the manufacturer's instructions.For viral nucleic acid detection in animal blood, as well as in the samples of BTV strains isolated previously (see Table F (5‐TAACGCAAAC GGAAATCGCT CA) and S2‐R (5‐GCGTTATCGG AAGATACCAA ACCA).For nucleic acid analysis of the BTV isolates from Shangri‐La, whole RNAs were extracted from BHK‐21 infected with the isolates and reverse transcribed to cDNA by SuperScript III (Thermo) with random primers. Seg2 fragments of 999 bp were amplified by Pfu (Tiangen) with primers S2‐2.2.4The PCR products of Seg1 and Seg2 were sent to the Kunming branch of the Tsingke Biological Technology Company for sequencing using primers Bs1p3f/Bs1p3r and S2‐F/S2‐R, respectively. Sequences in the middle of Seg1 (524–707 bp) and Seg2 (1654–2520 bp) were used for alignment.2.3Aedes Albopictus cell line C6/36 and baby hamster kidney cell line BHK‐21, in that order. The ECB virus isolation was performed as previously described  MEM (Gibco). The C6/36 cells were cultured at 28°C for 7 days. Then, 1 ml of the supernatant from C6/36 culture was inoculated into a T25 flask of BHK‐21 with 5 ml 2% FBS+ MEM. If CPE was observed, the flask with BHK‐21 was stored at 4°C until further analysis. If no CPE was observed, the culture was blind passaged up to three times before being called negative.The virus was isolated by the egg‐C6/36‐BHK‐21 (ECB) virus isolation system, in which the serum was inoculated in an embryonated egg, 2.42.4.1BTV (YN/2017)‐infected BHK‐21 cells (48 hpi) were collected by scraping and centrifugation. The cell pellet was suspended in 100 μl medium and lysed by freezing and thawing twice, followed by incubation with RNase A (Takara) at 37°C for 12 hr to delete cellular ssRNA  and 33.1A total of 507 yak serum samples were collected by the local ADCC during 2014 to 2017 in five villages within the Shangri‐La district. As shown in Table 3.2Culicoides, which is a critical factor in BTV transmission among ruminants.Three goat farms in one of the river valleys in Nixi  in Shangri‐La were selected for serologic investigation. The three farms were located on a hilltop, a hillside and the foot of the hill. Goats were investigated because they are popular ruminants raised in this rocky area. The serologic investigation by C‐ELISA suggested that there were notably high BTV infection rates (35.48%–65.63%) among the flocks of goats in the river valley  exhibited characteristics of BTV serotype 21.+ blood samples from 31 yaks and 10 sheep were collected from the highland area near the two surveillance sites in 2017 and tested by RT‐PCR. BTV nucleic acids were detected in the blood of 10 yaks and 2 sheep. For Seg1 fragment alignment, the samples from the positive yaks, positive sheep, 20 sentinel goats and four isolates from Shangri‐La and representative strains , also named BTV stain YN/2017, was sequenced completely. The Seg1 to Seg10 sequences of YN/2017 were registered in GenBank with accession numbers from MK250956 to MK250965.Online BLAST analysis (NCBI) for the complete sequences of 10 segments from YN/2017 showed that this BTV strain belongs to serotype 21 and is almost identical to three BTV strains  isolated in West Java in Indonesia in 1989–1990 Table . OverallFurthermore, 10 isolates from 10 sentinel goats were analysed. A group of Seg2 fragments with 999 bp were amplified by a Pfu PCR system with primers S2‐F and S2‐R. Eight of the PCR products with clear, bright target bands were sent for Sanger sequencing using primers S2‐F and S2‐R, while the other two PCR products with dim target bands were not sequenced. A multiple sequence alignment was performed for the eight confirmed DNA sequences with 867 bp (1654–2520 bp) and the corresponding Seg2 sequences of BTV strains YN/2017 and 5149E. The results showed that all 8 isolates had 100% identity with YN/2017 but showed apparent differences with 5149E (data not shown).4Thus far, a total of 14 serotypes of BTV  have been isolated in tropical and subtropical areas of China, and all serotypes except type 7 have been found in Yunnan Province  was sequenced completely. Interestingly, the genome of BTV strain YN/2017 was almost identical to those of three Indonesia BTV‐21 strains  were approved by their hosts, and the animal health was protected. The study was approved by the Ethics Committee of Yunnan Animal Science and Veterinary Institute. Click here for additional data file. Click here for additional data file.


The MR imaging and RF performance of the coil when switched between the 19F and 1H nuclei. When the coil was not switched to the resonance frequency of the respective nucleus being imaged, reductions in the transmit efficiency were observed of 32% at the 19F frequency and 12% at the 1H frequency. The coil provides transmit field homogeneity of ±12.9% at the 1H frequency and ±14.4% at the 19F frequency in phantoms representing the thorax with the air space of the lungs filled with perfluoropropane gas.The performance of the coil with MEMS or PIN diode switching was comparable in terms of RF measurements, transmit efficiency and image SNR on both 19F and 1H resonances were demonstrated, despite the proximity of their Larmor frequencies.MEMS and PIN diodes were found to provide comparable performance in on‐state configuration, while MEMS were more robust in off‐state high‐powered operation (>1 kW), providing higher isolation and requiring a lower DC switching voltage than is needed for reverse biasing of PIN diodes. In addition, clear benefits of switching between the  Trap circuits are commonly used to tune the coil resonance to multiple frequencies19F (60.06 MHz at 1.5 T) and 1H (63.8 MHz at 1.5 T) the bandwidth of passive traps with the typical Q‐factors of commercially available components is comparable to the frequency separation, limiting their use, as discussed previously.Q) of trap circuit inductors ,19F and 1H, respectively,19F and 1H when compared with respective single tuned coils. Therefore, due to the close frequencies of 19F and 1H at 1.5 T the use of switching is favored.In previous human lung imaging studies with perfluorinated Recent improvements in the technology for MEMS switches and associated driver circuitry has allowed increased switching speed, better power handling and reduced insertion loss,1H and 19F perfluorinated gases at 1.5 T.In this Note, we compare two methods for switching the matching network tuning: MEMS and PIN diodes, and compare these with a hard‐wired configuration for either nucleus. The switching comparison is exemplified using a four element fixed phase/amplitude transmit–receive RF coil designed for lung imaging of 22.1a and RFb in the circuit schematic model. More details on the device structure are provided in Keimel et al.The mechanism of actuating MEMS switches is fundamentally different to that of PIN diodes. The MEMS used here consist of an array of beam type structures that operate as relays actuated electrostatically by a DC voltage applied between the beam and gate.2.219F (60.06 MHz) and 1H (63.8 MHz) at 1.5 T on a GE Signa HDx system equipped with a 4 kW broadband RF amplifier. A circularly polarized B1+ excitation was achieved using a combination of 90° and 180° hybrid circuits that were custom built for both the 1H and 19F frequencies. In the matching network topography shown in Figure s was switched‐in to change the matching tuning (resonance) of the coil from the Larmor frequency of 1H to 19F by three possible mechanisms:To test the switching performance, a four element fixed phased transmit–receive coil was designed for dual tuned use for imaging (i) MEMS  switched on by application of 82 V DC,(ii) PIN diode  forward biased with 100 mA DC current,(iii) Hard‐wired configuration for either nucleus.Cm=68 pF  and Lm=92 nH for the left and right coils and Cm=56 pF and Lm=111 nH for the anterior and posterior coils, as annotated in Figure Cs=75 pF for all four coil elements. The manufactured matching networks are shown in Figure PLR, was calculated.The matching capacitance  and inductance values were; 3 volume. The widths were 25.5 cm for the anterior/posterior and 30 cm for the right/left elements coils. The coils were constructed from 11‐mm‐wide and 77‐μm‐thick self‐adhesive copper tape mounted on a flexible Polytetrafluoroethylene substrate. There were five capacitor break‐points in each coil.Decoupling between adjacent coil elements was achieved using capacitive decoupling networks.1H imaging and RF measurements a cylindrical phantom was used consisting of 3.6 g/L NaCl and 1.96 g/L CuSO4⋅5H2O salt solution19F imaging two glass canisters (2 L volume) were filled with C3F8 gas mixed with 21% O2 at 1.5 bar pressure, which emulates the air‐space in the human thorax. The glass phantoms were placed in a cylindrical shell and surrounded with a 12 L bag containing the saline solution and placed over another equal volume bag for suitable loading. The phantoms and coil are shown in Figure For 2.31 was performed in homogeneous phantoms by 2D spoiled gradient echo (SPGR) imaging. First, the flip angle (FA) was fit against image intensity with varying input power with TR >>T1 (TR = 600 ms for 1H phantom experiments and TR = 80 ms for 19F in the C3F8/O2 phantom). Next, T1 was fit against the image intensity with varying FA but with TR<T1 (TR = 8 ms for 1H phantom experiments and TR = 7.5 ms for 19F in C3F8/O2 phantom). Image intensity (SSPGR) was related to FA and T1 according to Deoniρ is a proportionality factor that depends upon system hardware and the TE, which was fixed. The noise standard deviation, σ, is included to normalize the signal to SNR units. The standard deviation of noise in images was measured in a signal‐free region of greater than 100 pixels as described in NEMA.Measurement of T1H and 19F SPGR imaging by varying input power and fitting FA, as well as ρ/σ, according to Eq. (1's for 1H and 19F (reported in results section) when the coil tuning was set to both 19F and 1H, respectively. The corresponding transmit efficiency with known input power and pulse width was subsequently calculated.To compare the effect of the respective switched tuning methods on transmit efficiency, the FAs were measured for g to Eq.  with the2.43F8 mixed with 21% O2 with a healthy adult male volunteer (28 years) following informed consent and a protocol approved by UK National research ethics committee. Three‐breaths of the gas were inhaled and then 3D 19F SPGR imaging was performed within a single breath‐hold (37 s scan time). In addition, 1H 3D SPGR anatomical imaging was performed during a separate breath‐hold (13 s) of air with the lungs at the same inflation level. Both images were localized to cover the same geometry. MEMS were used to switch between the two tuning states during in vivo imaging. A summary of all sequence and acquisition parameters used for the imaging experiments are provided in Table In vivo lung imaging evaluation was performed with inhaled C33.1Both the MEMS when switched closed, and PIN diode when forward biased, remained operational up to the maximum powers tested , in accordance with the maximum values specified in Supporting Information Table S1. However, as demonstrated in Figure Additionally, at high power there was an observed droop in the voltage over the pulse length when the diode was insufficiently reverse biased. This is believed to be related to rapid heating of the PIN diode during the RF pulse resulting in increased impedance. The waveform observed Figure D when th∼24 Ω for the anterior/posterior coils and ∼26 Ω for left/right coils . The 90 ° hybrid and 180 ° power dividers used had a reflection coefficient less than ‐15 dB for both frequencies with insertion loss of ∼0.5 dB. The decoupling between nearest neighbor coils  was optimized for the 19F frequency, where isolation was greater than 15 dB for quadrature channels. PLR was 12 ± 2% for the matching network for MEMS, PIN diode and hard‐wired configurations of the coil, which was verified with three repeated measurements.The measured unloaded and loaded quality factors of coils were 165 and 14.4 for anterior/posterior coils and 195 and 14.3 for right/left coils, respectively. The measured coil resistance when loaded with a cylindrical phantom was 3.21 of 1H in the salt solution phantom was 39.5 ms, while the T1 of 19F in the C3F8/O2 mixture was 16.6 ms. Using the FA mapping method described, the measured transmit efficiency within a cylindrical phantom at 63.8 MHz is shown in Figure 1H and 19F respectively. A measured reduction of ∼12% in the mean transmit efficiency and a ∼21% increase in the B1 inhomogeneity (SD) was observed when the resonance of coil was set to the 19F frequency, while transmitting and receiving at the 1H frequency. Similarly, for 19F, the mean transmit efficiency decreased by 32% and the B1 inhomogeneity (SD) increased by 67% when the resonance of coil was set to 1H.The measured Tρ/σ within the 19F phantom, as fitted from Eq.  and Sim4Life® (duke model)The measured transmit efficiency for the three switching methods are presented in Figure from Eq. , was 43.3.319F in vivo lung ventilation images overlaid on 1H images are shown in Figure 19F C3F8 lung ventilation MRI display similar SNR homogeneity to those performed with the phantom. SNR was found to be high enough (∼12) with the given imaging parameters for single breath‐hold lung ventilation images to be obtained and co‐registered with proton structural images.Eight central slices of 4PLR) when compared with a hard‐wired connection, as would be expected from their relatively low nominal ESR (Supporting Table S1). The loss in the matching networks is less than the insertion loss incurred across the power‐dividers used to feed power to the coil elements (0.3–0.5 dB loss for each stage) and primarily comes from the use of inductors, which have physically limited Q factors. There was only a 4.5% difference (10.6–11.1 μT/kW) in the mean transmit efficiency measured with the three switching configurations, which is likely in part due to the variation in re‐positioning the phantom. This is in accordance with other studies, which showed similar imaging performance with switched dual‐tuned coils when compared with single‐tuned counterparts.In this study, there was no measurable difference in the power loss introduced by MEMS or PIN diode switching  when coil tuned to 1H or switched to 19F tuning by the three methods: MEMS, PIN Diodes and hard‐wiredFIGURE S2 (A) Simulated transmit efficiency at 63.8 MHz within a cylindrical phantom in central axial and coronal slices. Simulated transmit efficiency (µT/kW) with 1 kW RMS input power at 60 MHz using HFSS (B) or SIM4LIFE (C) using realistic human body models. The mean transmit efficiency ± standard deviation shown above axial slices is calculated within the volume of the circled region with phantom and over the displayed region in human body models. Greater inhomogeneity is observed in HFSS human model due to the larger size, thereby having regions much closer to conducing elements of coil. However, Local 10g averaged SAR for the same input power calculated by HFSS or SIM4LIFE with the body models at 60MHz were close at 121 W/kg and 125 W/kg, respectivelyTABLE S1 Performance parameters of common switching devices: MEMS, PIN diodes, and FETs.Click here for additional data file.