Eto, of 3.60 V μm−1 and a good stability. Further, the dependence of (turn‐on field) Eto/(threshold field) Ethr and effective work function, Φe, on temperature is investigated and the possible mechanism of temperature‐dependent field emission phenomenon has been discussed. Moreover, electronic transport in a single UBNS reveals it to be an intrinsic p‐type semiconductor behavior with carrier mobility about 1.26 × 10−1 cm2 V−1 s−1, which is the best data in reported works. Interestingly, a multiconductive mechanism coexisting phenomenon has been explored based on the study of temperature‐dependent conductivity behavior of the UBNSs. Besides, the photodetector device fabricated from single‐crystalline UBNS demonstrates good sensitivity, reliable stability, and fast response, obviously superior to other reported boron nanomaterials. Such superior electronic‐optical performances are originated from the high quality of single crystal and large specific surface area of the UBNSs, suggesting the potential applications of the UBNSs in field‐emitters, interconnects, integrated circuits, and optoelectronic devices.Large‐scale single‐crystalline ultrathin boron nanosheets  are fabricated through an effective vapor–solid process via thermal decomposition of diborane. The UBNSs have obvious advantages over thicker boron nanomaterials in many aspects. Specifically, the UBNSs demonstrate excellent field emission performances with a low turn‐on field,  Furthermore, the temperature‐dependent FE measurements demonstrated that the temperatures greatly affect the turn‐on or threshold electric fields, emission current densities, and the effective work functions. Moreover, the field‐effect transistor (FET) measurement reveals that the UBNSs are p‐type semiconductors with carrier mobility of 1.26 × 10−1 cm2 V−1 s−1, two orders of magnitudes larger than current reported data.5%, and higher specific detectivity of 4.91 × 1011 Jones,[[qv: 16a]] compared to reported boron nanomaterials. These results suggest that the UBNSs are promising candidates to apply in both high performance field emission electron source and high‐speed photodetectors/photo‐electronic switches with high sensitivity.In this paper, single‐crystalline UBNSs were synthesized by an effective vapor–solid (VS) process via thermal decomposition of diborane  and (200) crystal faces of α‐tetragonal boron (JCPDS No. 75‐0218). Figure The detailed crystalline microstructure of the UBNSs was investigated by transmission electron microscopy (TEM). The low‐magnification TEM image of a typical individual NS is shown in 2.2FigureΦ value is deduced to be 4.81 eV for the UBNSs , which is very close to boron bulk materials and nanostructures.[[qv: 12e]],The ultrathin structure and good crystallinity suggest that the UBNSs are a potentially excellent field‐emission cathode. Interestingly, to the best of our knowledge, this work is the first study on boron nanosheet‐based field emission. We have employed ultraviolet photoemission spectroscopy (UPS) to measure the work function of the UBNSs. The full He I scan of the UPS spectrum of the films is shown in −7 Pa at RT (293 K). The evolution of field emission current density (J) with applied electric field (E) has been shown in Figure −2) at a macroscopic field of as low as 3.60 V μm−1 and a high FE current density of 7.47 mA cm−2 at a macroscopic field of 5.80 V μm−1 can be read from Figure 6/NdB6 nanorods,4C nanowires,2 sheets,In order to further explore the feasibility of applying the UBNSs in the vacuum microelectronic and nanoelectronic fields, the field‐emission characteristics were measured under a pressure of 8.0 × 10 at RT 29 K. The e at RT 29 K. The eA and B are the constants with values of 1.54 × 10−6 A eV V−2 and 6.83 × 103 eV−3/2 V μm−1, respectively; J, E, and β represent the current density, the applied field, and the field enhancement factor, respectively; Φ is the work function of the material (4.81 eV for the UBNSs). Generally, the β are usually associated with the emitter geometry, alignment, crystal structure, and spatial orientation of distribution of emitting centers. For instance, the FE performance of LaB6 nanowires has been proved to be dramatically improved by sharpening the emitter tip, increasing their aspect ratios, spatial uniformity, and alignment .5–106). Moreover, the FE stability measurements of the UBNSs  Equation Figure−1, the threshold fields (at an emission current density of 1 mA cm−2) reduce dramatically from 5.08 V μm−1 to 3.72 V μm−1, whereas the emission current density rises remarkably from 1.24 to 5.77 mA cm−2  upon increasing the temperatures from RT to 573 K, as listed in TableJ/2E) versus 1/E, are depicted in Figure Φ) and field‐enhancement factor (β).β (1363) exhibits a neglectable fluctuation with temperature; therefore, the effective work function (Φe) under different temperatures can be deduced from the relation Tk = −BΦe3/2/β (Tk is the F–N slope at temperature T).Φe) decreases from 4.81 to 2.35 eV with the increase of temperature, which explains well the obvious decrease of the turn‐on field and the significant increase of emission current with temperature increasing.Temperature of the cathode has a great impact on the FE characteristics of material.EF) with temperature lowers the effective work function (Φe).[[qv: 32a]],Φe is increasing from 4.81 to 4.83 eV , which contradicts with the FE data shown in the inset of Figure β) lowers the effective work function.[[qv: 32b]],β, so the enhancement factor should be a constant.Figure2 and N2, etc.) adsorbed on the UBNSs surface capture free electrons from the UBNSs, which forms a depletion layer on the UBNSs surface  and carrier mobility (≈102 cm2 V−1s−1); however, previous reported results showed much lower conductivity (10−3−10−7 Ω−1 cm−1),−2−10−8 cm2 V−1 s−1)−2 Ω−1 cm−1 in vacuum at RT , which is the highest value among reported results of pure bulk boron, boron nanowires, and nanobelts devices.2 on the top  of FET at VSD = 2 V is depicted in Figure μ = 1.26 × 10−1 cm2 V−1 s−1 . This observed mobility is about two orders of magnitudes of that of nanowires, and also higher than that of pure bulk boron.Boron has a theoretical high conductivity –1000/T, the whole temperature range can be divided into two temperature regions: a high‐temperature region (100 K < T < 600 K) and a low‐temperature region (20 K < T < 100 K). In the high T region, the measured conductivity σ of the UBNSs as a function of 1000/T is shown in Figure σ1 and σ2 are the prefactors of conductance, E1 and E2 are two kinds of activation energies during the thermal activation conduction processes. The red line in Figure E1 (233 meV) mode and E2 (17 meV) mode are related to the deep acceptor states and the shallow acceptor states, respectively. No intentional doping for holes is carried out in our experiments; thus, the origin of holes is unclear at present. One probable explanation might be related to the excess oxygen,[[qv: 16a]] which has two possible models: one is cation vacancy and the other is interstitial oxygen. Oxygen playing an important role in the origin of positive holes can also be found in other p‐type semiconductors, such as CuAlO22O films.In order to investigate the transport mechanism of the UBNSs, the DC resistivity of single UBNS was measured under different temperatures. The insets of T region, the estimated activation energy is around 0.8 meV if the experimental data (20 K < T < 100 K) are also fitted with the Arrhenius law. However, the fitted activation energy is unreasonably low, which indicates that the conductivity of UBNSs at the low‐temperature region is dominated by a complete different conduction transport mechanism. It is generally accepted that at a low temperature region, temperature‐dependent conductivity follows the Mott variable‐range hopping (VRH) modelσ0 is the prefactor of conductance and T0 is a constant. Figure σ) and (1/T)1/4 in the low‐temperature region (20 K < T < 100 K), and the red line in Figure T < 600 K) and Mott's variable‐range hopping mechanism dominates the low‐temperature region (20 K < T < 100 K). Actually, such a manifestation (multiple conduction mechanisms coexistence) was also discovered in other materials such as V2O5 nanowires,2 films,In the low‐Figure2/Si substrate by using a standard lithography procedure. The uncovered part of the nanosheet, i.e., channel of device was exposed to the incident light. The SEM image of the single UBNS‐based device is shown in the upper inset of Figure I–V curves of a single device, measured under the dark condition and under 325 nm light illumination (17.9 mW cm−2), are presented in Figure I–V curves. Different from Schottky contact that is usually observed in nanoparticle/nanowire‐based photodetectors,The high crystallinity of the UBNSs, combined with a band gap of 1.5 eV, benefits the UBNSs to be applied in optoelectronics and microelectronics. As illustrated in the bottom inset of λR, which evaluates the response of the photocurrent to incident optical power on the active area; 2) external quantum efficiency EQE, which calculates the numbers of electron–hole pairs detected per incident photon; 3) specific detectivity D*, one of the typical values used to evaluate the performance of a detector, which measures a photodetector using the unit of cm Hz1/2 W−1 . Based on the present experimental results, the values of λR, EQE, and D* of the UBNSs are 465 A W−1, 1.78 × 105%, and 4.91 × 1011 Jones, respectively . These values are the first report for boron nanostructure‐based nanodevices and also much higher/or comparable to other semiconductor photodetectors.[[qv: 55a]],Besides the response time, there are other three critical parameters for the evaluation of a photodetector performance. 1) Responsivity 3−1, a low threshold electric field of 5.08 V μm−1, a high electric field enhancement factor of 1363, and a high current density of 7.47 mA cm−2 at 5.80 V μm−1. Moreover, the FET measurement demonstrated that the UBNSs showed an intrinsic p‐type semiconductor behavior with the carrier mobility of 1.26 × 10−1 cm2 V−1 s−1. The electronic transport can be explained by double thermal activation processes at high temperature (100 K < T < 600 K) and Mott's variable‐range hopping process at low temperature (20 K < T < 100 K). In addition, the UBNSs exhibit a fast, reversible, and stable photoresponse. Besides, a high photoconductive gain (465 A W−1), a high quantum efficiency of 1.78 × 105%, and a high specific detectivity of 4.91 × 1011 Jones were also observed. The high photosensitivity and fast photoresponse result from the high crystallinity of the UBNSs, large specific surface area exposed to light, free of trap states, and lower recombination barriers in the single‐crystalline UBNSs. These results reveal that the UBNSs have great potential applications in electronics and optoelectronics.In summary, large‐scale and high‐quality single‐crystalline UBNSs were fabricated through an improved VS process method. HRTEM and SAED show that the boron nanosheets have an α‐tetragonal structure with good crystallinity orientated along the [002] direction. Field emission properties of the UBNSs have been investigated first time, which showed a low turn‐on electric field of 3.60 V μm4Synthesis and Characterization of UBNSs: The synthesis of UBNSs was conducted by a catalyst‐free VS process in a 30 mm external‐diameter quartz tube placed inside as shown in Figure S3 (see the Supporting Information). In brief, several pieces of cleaned silicon wafers were located at upstream as substrates for deposition. Before the heating process, the quartz tubes were first pumped to low pressure ≈0.5 Pa and then washed with a mixed gas , and this process was repeated for three times to remove oxygen in the system completely. Then, the system was heated to a target temperature, 950 °C, with a temperature rising rate of ≈15 °C min−1 under pumping vacuum. A steady mixed gas (5% B2H6 + 95% Ar) with a flow rate of 5 sccm was introduced into the system for 120 min, and then the reaction pressure indicated as ≈8 Pa. After that, the furnace was powered off and the temperature was dropped down to RT naturally.−6 Pa. The field‐emission properties were conducted at different temperatures based on a home‐built parallel‐plate FE system in a system (8.0 × 10−7 Pa).The UBNSs were characterized by a field‐emission scanning electron microscope , a transmission electron microscopy , and an atomic force microscopy . The Raman spectra were recorded by a laser Raman spectrometer  using 532 nm Ar ion laser as the excitation source. Ultraviolet photoelectron spectroscopy (UPS) was measured using an ESCALab 250Xi from Thermo Scientific under a vacuum of ≈6.0 × 10Device Fabrication and Characterization: To fabricate a single‐nanostructure device, the UBNSs were dispersed in ethanol and then deposited on the surface of SiO2 (500 nm)/Si(P+). The standard photolithography technique  followed by Cr/Au (10 nm/100 nm) electrodes deposition with EB evaporation  and a standard lift‐off procedure were adopted here. The current–voltage characteristics were monitored by a low‐temperature cryogenic probe station  and a semiconductor parameter analyzer .For photodetector measurement, all the measurements were conducted in atmospheric conditions and the photocurrent was collected by a semiconductor parameter analyzer . A He–Cd laser  was focused and guided on the NS device perpendicularly. To measure the time response of the B NS device to light irradiation, a mechanical chopper was used to turn on and off the light irradiation. The light intensity was calibrated by using a UV‐enhanced Si photodiode.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The wavelengths contained in a revised list of 4He I lines from 320 A to 21132 A are also based on the best measurements, over half of them having been calculated from the wavenumber separations of appropriate levels. Several previously disturbing features of the 4He term scheme are obviated by the revised level values. In a discussion of the experimental results for this spectrum some comparisons with theory are made.A table of energy levels based on the most accurate observations now available is given for the neutral  A Multiplet Table of Astrophysical Interest and An Ultraviolet Multiplet Table [Atomic Energy Levels [It seems important that an up-to-date helium line list and a table of revised energy levels be made generally available. The use of helium in many kinds of spectroscopic sources, in studies of electrical discharge mechanisms and of plasma conditions, and the importance of helium in astrophysics combine to make He I one of the spectra most frequently observed. The most recent essentially complete compilation of helium wavelengths, contained in et Table ,1 appearet Table , 3 in haet Table  recent oy Levels , have le4He and 3He) illustrates the importance of maintaining as accurate data on the helium-type atoms as contemporary experimental techniques allow.In pointing out the need for the present revision of the He I level values, it may be well to emphasize the special place of two-electron atoms in atomic theory. Since these are the simplest atoms for which exact calculations in either nonrelativistic or Dirac's relativistic wave mechanics cannot at present be made, they furnish excellent testing grounds for the various approximation methods of quantum mechanics and theories of the interaction between electrons. Indeed, until the recent beautiful measurements by Herzberg  the theoHandbuch der Spectroscopie [4He I to date.Naturally, the present discussion of experimental results will stress the more recent observations. The references are mainly to work done since the appearance of volume VII of the roscopie . Since t3He I spectrum. Because of the large percentage mass difference of 3He and 4He and because 3He has a nonzero nuclear magnetic moment giving rise to hyperfine structure, the 3He I spectrum differs in detail a good deal from 4He I. A reasonably comprehensive article on the experimental results for 3He I would be about as long as this one. Perhaps one of the investigators of that spectrum will supply such an article.No attempt has been made in this paper to deal with the interesting and important Except for a few normally very weak “forbidden” lines, s2s3S—2s2p3P°, is open to objection since the upper term is subject to autoionization into the 3P° continuum above the 1s∞p series limit. A weaker line observed by Kruger [The wavelengths of the vacuum ultraviolet lines were calculated from adopted level values except for the line at 320.392 A. Wu  has madey Kruger  at 357.5y Kruger  and Holøs2 1S0—np1P°1 to n=15, as well as the forbidden transitions 1s2 1S0—ns1S0 to n = 7, 1s2 1S0—2p, 3p3P°1, and 1s2 1S0—3d1D2. Bomke [s2 1S0 as 198307.9 cm−1 from Kruger’s measurements. From this adopted value for the ground term he then calculated wavelengths for the 1s2 1S0—np1P°1 series to n=2. Using this procedure, which is essentially that followed here, Boyce and Robinson [The original investigation of the helium spectrum in the vacuum ultraviolet was made by Lyman , who dis2. Bomke  had earl2. Bomke , of Krug2. Bomke ). PascheRobinson  obtainedThe earlier wavelength measurements made in the vacuum ultraviolet region are, of course, superseded by the work of Herzberg  and by tThe intensities for the vacuum ultraviolet lines in wavenumber which is the primary adopted quantity, and the wavelength is taken from the Edlén formula to the number of figures regarded as significant.Care has been taken to assure that the wavelengths given for lines in the air region should also be the best values now available. Almost all of the 192 lines given for this region have been observed, but wavelengths calculated from adopted level differences are considered more accurate than observed values for about half of these. Most of the wavelengths which are taken directly from observation also correspond exactly to the level differences involved, since they were used to determine these differences. Besides the 100 calculated air wavelengths, the table includes 27 other new air values; these are mostly either unpublished measurements by Shenstone  or weighOnly two “forbidden” lines in the air region are- given in Normally unobserved forbidden transitions in helium appear quite readily when the radiating gas is in a strong electric field. Investigations of the Stark effect in He I probably culminated in the beautiful work of Foster , 20, whojg-value for 2s3S1 to an accuracy of 1 ppm. by Drake et al. [Most investigations of the Zeeman effect in He I carried out in the last 30 years have been concerned with forbidden lines. The work of J. Brochard and P. Jacquinot  is rathee et al.  using miThe quantitative intensity measurements of Crosswhite and Dieke  show strC. J. Humphreys  has veryIt is hoped that the intensities given in 4He I spectrum. The first step in obtaining revised energy level values  was to fix the d3D—3d1D separation, and it is unlikely that the intersystem connection is in error by more than 0.02 cm−1. A recent unpublished interferometric measment of −1) has been combined with the average of two recent interferometric determinations of −1. Herzberg’s estimated uncertainty in the wavenumbers of the observed lines is +0.15 cm−1.A new square array has been made for the surement  of the 3p3P° intervals as 2—1 = 0.02 cm−1 and 1—0 = 0.27 cm−1. He had not resolved the smaller separation, but guessed at it from the analogy with the 2p3P° term and his measurement of the −1, respectively. These results proved in disagreement with theoretical expectations [n. However, only in 1951 did Fred et al. [p3P° intervals given in p and 3p triplet levels are given to four decimal places in The measurements used here to determine the resolved fine structure intervals deserve some discussion, since all of these have been improved in the last few years. The d et al. , while tand Lamb . are not accurately enough measured to check on the constancy of the interval ratio given in the table, and the values given can only be taken as indicative. Brochard et al. [4He I fine-structure intervals with the results of various theoretical calculationsTheoretical calculations of by Breit  and Arakby Breit . A more by Breit  predictsd et al.  compare f1F° and 4f3F° terms are based primarily on Mlle. Pilon’s measurements [d1D—4f1F° and 4d3D— 4f3F° separations. To a lesser extent they are also based on the measurements by C. J. Humphreys and H. J. Kostkowski [f terms to 3d terms. The agreement is within the expected error of Humphreys and Kostkowski, and the 5f terms positions given here are based entirely on their measurements.The new positions given in urements  of the 4stkowski  of the ip1P° level from terms of every possible L-value belonging to the configurations 1s 5l, 6l, and 7l. Since his measurements were made in rather strong electric fields, they cannot be used directly to obtain unperturbed energy levels. From a comparison of Foster’s estimated wavelengths for certain transitions at zero field with those calculated from the 6f and 7f term values in L from one of these lines whose zero-field wavenumber was known. These separations could be corrected to zero-field strength by comparing Foster’s nd1D—1P° separation with the known zero-field separation of these levels for each n. The term positions were also based on values found from plotting the very regular Rydberg denominators, particularly in the case of the 7l terms. In this manner the positions of terms belonging to the 5g, 6g, 6h, 7g, and 7h configurations have been calculated and included in In his investigations of the Stark effect in He I, J. S. Foster  observednf singlet series members falling below the corresponding triplets from n = 5 on.  Similarly, the earlier data indicated that the nd1D series crossed over and fell below the nd3D series at n = 9. It is now clear that this puzzling behavior was not real. Shenstone’s much improved measurements [nd triplet and singlet series from 8d on showed that the apparent crossing-over of those series at 9d was due to inaccurate observations. His measurements were essentially confirmed and the singlet series extended to 18d by Herzberg [−1 relative to the triplets, has removed the slight crossing- over at 13d which remained after Shenstone’s work. This accurate connection is also mainly responsible for rendering the nf series quite regular with the singlets probably slightly above the triplets. The observations are, however, not sufficiently accurate above 4f to fix the relative positions of the terms. One can say, however, that beyond 5f the 1F° terms are probably not separated from the corresponding 3F° terms by more than 0.1 cm−1.New level values based on recent improved series measurements and the accurate connection between the singlet and triplet systems have obviated several features of the He-atom energy level scheme which were previously disturbing. The best previous list of helium energy levels stkowski  listed burements  of the nHerzberg . The newnp1P° from 11p on are taken from a series formula. Paschen and Götze apparently thought this method would give better values than any measurements available to them; since there have been no new observations, the series has been recalculated to 20p. Higher members of the 3P° series were obtained by converting wavelengths from Paschen-Götze to vacuum wavenumbers according to Edlén’s tables. Several numerical errors occurring in Paschen-Götze have been corrected.The energies for members of the series Combining the limits obtained by Herzberg  for thred3D—nf3F°:p3p°2,1—nd3D:p1P°1—nd1D2:From the series 3−1, is in excellent agreement with the experimental ionization energy of 4He adopted by Herzberg in his paper, 198310.82±0.15 cm−1. The spread in the three values obtained here is reduced from that obtained by Herzberg, no doubt because the available data have already been averaged once in obtaining values for the fixed term of each series.The average of these values, 198310.81 cmIn conclusion, it might be mentioned that the excellent article by Bethe and Salpeter  on the t


Leishmania is an early‐branching eukaryote with a remarkably plastic genome, which hints at peculiar genome maintenance mechanisms. Here, we investigated the existence of homologs of the 9‐1‐1 complex subunits in L. major and found that LmRad9 and LmRad1 associate with chromatin in response to replication stress and form a complex in vivo with LmHus1. Similar to LmHus1, LmRad9 participates in telomere homeostasis and in the response to both replication stress and double strand breaks. However, LmRad9 and LmHus1‐deficient cells present markedly opposite phenotypes, which suggest their functional compartmentalization. We show that some of the cellular pool of LmRad9 forms an alternative complex and that some of LmHus1 exists as a monomer. We propose that the diverse assembly of the Leishmania 9‐1‐1 subunits mediates functional compartmentalization, which has a direct impact on the response to genotoxic stress.The Rad9‐Rad1‐Hus1 (9‐1‐1) complex is a key component in the coordination of DNA damage sensing, cell cycle progression and DNA repair pathways in eukaryotic cells. This PCNA‐related trimer is loaded onto RPA‐coated single stranded DNA and interacts with ATR kinase to mediate effective checkpoint signaling to halt the cell cycle and to promote DNA repair. Beyond these core activities, mounting evidence suggests that a broader range of functions can be provided by 9‐1‐1 structural diversification. The protozoan parasite  Thus, an extensive network of pathways coordinates DNA damage sensing, cell cycle progression and DNA repair processes. The Rad9‐Rad1‐Hus1 (9‐1‐1) heterotrimeric complex is a central player in the DNA Damage Response (DDR) of eukaryotic cells. The ring‐shaped 9‐1‐1 complex is structurally related to the PCNA clamp that acts in DNA replication and is loaded onto DNA during the early steps of the DDR  ends  that presents ∼21% identity with the human Rad1 at the primary sequence level, and is phylogenetically related to Rad1 homologs from other eukaryotes . As presented in Fig. We have previously reported that L. major 9‐1‐1 complex, we initially performed a pull‐down assay, using an in vitro translated hemagglutinin‐tagged version of LmRad1 (HA‐LmRad1) as bait. The western blot analysis presented in Fig. To investigate if LmRad1 could be part of a in vivo we performed co‐immunoprecipitation assays (coIP) using a cell line expressing a C‐terminally 12xMyc tagged version of LmHus1 (LmHus1‐12xMyc) . We used anti‐Myc antibody to immunoprecipitate LmHus1‐12xMyc and, as presented in Fig. in vivo.To further test the association of LmRad1 with 9‐1‐1 subunits et al., in vivo , which also carries pXG1‐LmRad9, but in which both genomic alleles of LmRad9 are intact. As presented in Fig. Rad9 cells had no detectable pXG1‐LmRad9 molecules after 30 passages (∼200 cell divisions) as monitored by semi‐quantitative PCR analysis. In contrast, the LmRad9−/−/+ cells did not show any significant loss of the pXG1‐LmRad9 signal over the same number of passages, which suggests that LmRad9 is essential for survival.We have previously reported that LmHus1 deficiency interferes with the maintenance of telomeres and impairs a proper response to genotoxic stress . However, the level of EdU incorporation in individual cells varied significantly, as shown in Fig. Based on the results described above, we decided to investigate whether the distinct effects of LmRad9 and LmHus1 deficiencies are limited to the replication stress response or characterize a general response to DNA damage. Thus, we analyzed the recovery profile of LmRad9+/− and LmHus1+/− cells after exposure to the radiomimetic drug phleomycin (phleo), which causes a high frequency of DSBs in DNA Moore, . ContrarWe next asked if the marked differences in the proliferation rates between the two cell lines upon phleo exposure would correlate with alterations in the cell cycle profile. Thus, we again used flow cytometry analysis to assess DNA content in the LmRad9+/− and LmHus1+/− cells cultivated in the presence of phleo. As shown in Fig. Trypanosoma brucei equivalent of γH2AX  and LmHus1 (∼37 kDa) have a coincident elution peak (fractions 57–68) with an apparent molecular mass between 150 and 200 kDa  and Mcm10, a component of the pre‐replication complex  bearing LmRad9 (and lacking LmHus1 and LmRad1) and monomeric LmHus1, as shown in the tentative model presented in Fig. et al., et al., et al., Despite the novelty of the observations that we make here, it is noteworthy that most studies assume that the effect of the individual abrogation of Rad9, Rad1 or Hus1 expression is a consequence of the disruption of the entire 9‐1‐1 complex, without taking into account the possible existence of alternative complexes. Indeed, the dissection of functional compartmentalization of individual subunits is problematic due to dual functions within and outwith the 9‐1‐1 complex. Thus, it is possible that the phenotypes that we observe for LmRad9 and LmHus1 are detectable because of pronounced, lineage‐specific roles for the subunits outwith the Leishmania 9‐1‐1 homolog subunits play a more widespread role in DNA metabolism than might have been anticipated. The separation of function between LmRad9 and LmHus1 seems to be based on the formation of at least one noncanonical LmRad9‐containing complex, and may reveal novel activities of monomeric Hus1. Further studies are required to dissect the activities of LmRad9 and LmHus1 and to evaluate their participation in key events that shape the biology of Leishmania and related protozoa.In summary, our data indicate that the L. major LT252 (MHOM/IR/1983/IR) and transfected cell lines derived from this strain were cultured as promastigotes in M199 medium containing 10% heat‐inactivated fetal bovine serum at 26°C. The heterozygous or episome‐bearing cell lines were generated using the transfection protocol previously described ; serum was further affinity‐purified using the recombinant protein as bait. The chicken anti‐LmRad1 antibody was raised against recombinant 6xHis‐LmRad1expressed in E. coli BL21 cells from the pET28aLmjF.20.0390 construct, and purified by ammonium sulfate precipitation and ion exchange chromatography. The mouse anti‐LmRad1 serum was also raised against recombinant 6xHis‐LmRad1. The anti‐γH2A antibody was generated by immunizing rabbit with the phospho‐peptide KHAKA[pT]PSV ; serum was further affinity‐purified using the corresponding peptide as bait. The commercial antibodies used were as follows: anti‐Myc and anti‐EF1α ; anti‐H2A ; anti‐HA . For western blotting analysis, proteins were resolved in SDS‐PAGE, transferred to PVDF membrane and analyzed with the indicated antibodies. Bands were detected with ECL Prime Western Blotting Detection Reagent  and visualized with Hyperfilm ECL (GE Life Sciences) or ImageQuant LAS 4000 (GE Life Sciences). For data in Figs Rabbit anti‐LmHus1 and anti‐LmRad9 antibodies were previously described . After brief sonication, extracts were clarified by centrifugation  and incubated with immobilized HA‐LmRad1 for ∼2 h at 4°C under agitation. Beads were washed with pull down buffer and analyzed by western blotting.LmjF.20.0390 ORF was cloned into pTCFE1‐NHA vector. The resulting pTCFE1‐NHA‐LmRad1 construct was used with the 1‐Step Human‐Coupled IVT Kit (Thermo Scientific), according to manufacturer instructions, to generate the HA‐LmRad1 fusion. HA‐LmRad1 was immobilized with protein A‐agarose beads coupled with anti‐HA antibody. Total cell extracts from 9 cells were harvested and lyzed in ice cold Buffer A . After clarification with centrifugation , extracts were incubated for ∼4 h at 4°C with Dynabeads M‐280 Sheep Anti‐Mouse IgG beads (Thermo Fisher Scientific) previously coupled with either mouse anti‐LmRad1 serum or mouse anti‐Myc monoclonal antibody. Beads were collected with a magnetic rack and washed 3× in Buffer A. CoIP products were suspended in Laemmli and analyzed by western blotting.For co‐immunoprecipitation (coIP) ∼10et al., 7 cells were harvested, washed with 1xPBS and incubated with extraction buffer  for 10 min on ice. The suspension was centrifuged  and the supernatant was saved as soluble fraction (Soluble I). The precipitated insoluble material was treated with extraction buffer again, centrifuged as before and the supernatant was saved as soluble fraction (Soluble II). The pellets were treated with DNaseI Amplification Grade  (10 units for 5 × 107 cells) for 20 min at room temperature. The sample was centrifuged , and the supernatant was saved as DNaseI released fraction (Chromatin).Soluble and chromatin bound proteins were fractionated as described before . Lysis was performed on ice for 30 min. Extracts were clarified by ultracentrifugation . Supernatant was applied to a Superdex 200 column  previously equilibrated with lysis buffer and connected to ÄKTA purifier system (GE Life Sciences). Lysate was resolved with a flow rate of 750 μl per minute and 1 ml fractions were collected.Soluble extracts were prepared from ∼5 × 10Cells were harvested, washed with 1X PBS and fixed in 30% PBS/70% methanol overnight at 4°C. Fixed cells were washed with 1X PBS and stained in 1X PBS containing Propidium Iodide 50 µg/ml) and RNase A (100 µg/ml) at 37°C for 20 min. Flow cytometry data was collected using a BD FACSCanto flow cytometer  for 2 h. Cells were fixed with 3.7% Formaldehyde for 15 min and then adhered into poly‐L‐lysine coated slides. Cells were permeabilized with 0.5% TritonX100 for 20 min, washed three times with PBS‐3% BSA and then incubated with the Click‐iT reaction cocktail for 30 min at room temperature. DNA was stained with Hoechst 33342. Images were acquired with a LSM 780 Axio Observer microscope . Quantification of EdU fluorescence intensity was performed with ImageJ software.[JDD] planned and performed the experiments and wrote the manuscript; [RO] did the Southern blot presented on supplementary Supporting Information Figure S3, the PCR on Fig. Supporting Information.Click here for additional data file.


However, EPI suffers from Nyquist ghosting, which normally is corrected for by acquiring a reference scan. In a dynamic acquisition of a series of images, this results in the sacrifice of a time point if the reference scan involves a full readout train with no phase encoding. This time penalty is negligible if an integrated navigator echo is used, but at the cost of a lower signal‐to‐noise ratio (SNR) as a result of prolonged T2* decay. We describe here a workflow for hyperpolarized 13C EPI that requires no reference scan. This involves the selection of a ghost‐containing background from a 13C image of a single metabolite at a single time point, the identification of phase correction coefficients that minimize signal in the selected area, and the application of these coefficients to images acquired at all time points and from all metabolites. The workflow was compared in phantom experiments with phase correction using a 13C reference scan, and yielded similar results in situations with a regular field of view (FOV), a restricted FOV and where there were multiple signal sources. When compared with alternative phase correction methods, the workflow showed an SNR benefit relative to integrated 13C reference echoes (>15%) or better ghost removal relative to a 1H reference scan. The residual ghosting in a slightly de‐shimmed B0 field was 1.6% using the proposed workflow and 3.8% using a 1H reference scan. The workflow was implemented with a series of dynamically acquired hyperpolarized [1‐13C]pyruvate and [1‐13C]lactate images in vivo, resulting in images with no observable ghosting and which were quantitatively similar to images corrected using a 13C reference scan.Single‐shot echo planar imaging (EPI), which allows an image to be acquired using a single excitation pulse, is used widely for imaging the metabolism of hyperpolarized  Alternatively, a 1H reference image can be acquired before the hyperpolarized 13C image13C reference echoes can be acquired by obtaining extra k‐space lines without phase encoding.in vivo images.Nyquist ghosting in hyperpolarized 2x axis). The overall phase error is:B0 shift induced by a gradient pulse) and x direction. The phase error accumulated as a result of a group delay is equivalent to the effect of a first‐order short time constant eddy current in the readout gradient and is also included in Equation k‐space lines, resulting in Nyquist ghostingρ^ is the reconstructed image, N is the number of phase‐encoding steps and x/y are the coordinates in the ideal image in the frequency‐ and phase‐encoding directions, respectively. In short, the ideal image splits into a real one, at the original location, and an imaginary one, half a FOV away. This model typically holds for images acquired from non‐oblique planes on modern MRI systems, where cross‐term and higher order term eddy currents are limited.Half‐field of view (FOV) ghosting in EPI images is induced by: 1) group delays between readout gradients and signal acquisition; and 2) eddy currents in the readout direction.13C signal, which was obtained from the proton image. The proposed workflow is illustrated in Figure A reference scan can be used to estimate 2.113C reference scan13C reference echoes1H reference scan.13C imaging sequence acquired a 2D EPI k‐space, starting with an excitation pulse and slice selection gradient, followed by a train of 20 bipolar readout gradients and, simultaneously, a train of 19 blipped phase‐encoding gradients. The acquisition matrix was 32 × 32, but only four k‐space lines were acquired before the centre of k‐space in order to minimize the echo time (TE) and enhance the SNR; hence, only 20 bipolar readout gradients were used (acquiring 62.5% of k‐space). For the images acquired in vivo, a 15.936‐ms spectral‐spatial (SpSp) pulse  was used for excitation, whereas, for imaging phantoms, a 4‐ms sinc excitation pulse with a bandwidth of 1600 Hz was used. The sinc pulse was designed in Matlab  using the SLR algorithmhttp://rsl.stanford.edu/research/software.html). The envelope‐pulse and subpulses in the SpSp pulse were also generated using this toolbox, and the envelope‐pulse was then discretely sampled by the subpulse to achieve the desired stopbands between replicated excitation bands. The 13C reference scan was acquired with the same imaging parameters, but with the phase‐encoding gradients turned off. For 13C imaging with integrated 13C reference echoes, three extra readout lobes, with no phase‐encoding gradients, were inserted in front of the regular readout train. Each of the inserted lobes had the same shape as the regular readout lobe. For the 1H reference scan, the same excitation pulse (same pulse width and flip angle) and the same readout gradient train (same amplitude and timing) were used as for 13C imaging, but with the phase‐encoding gradients turned off. The FOV in the readout direction of the 1H reference scan was therefore approximately one‐quarter (13C image. The slice selection gradient for the 1H reference scan was reduced from that used for 13C imaging to ensure the selection of the same slice.The proposed workflow was compared with three other phase correction methods: 1) direct 2.21H/13C transmit volume coil, 1H receive volume coil and a 20‐mm‐diameter 13C surface receive coil . The bore size of the gradient coil was 120 mm. The maximum gradient strength was 0.4 T/m and the maximum slew rate was 3000 T/m/s.Phantom experiments were performed on a 7‐T Agilent scanner  with a 13C sinc pulse was used in the 13C imaging sequence to excite a 10‐mm slice. Proton fast spin echo (FSE) images were acquired with a 256 × 256 matrix from the same slices and same FOVs as used for 13C imaging, in order to provide a positional reference. The proposed workflow was compared with the other methods for phase correction under a variety of different acquisition conditions. The receiver bandwidth and echo spacing are given below. The former affects the phase error accumulation and the latter determines image distortion.A 90° flip angle 2.2.113C]lactate with a 4 × 4‐cm2 FOV, receiver bandwidth of 125 kHz  and echo spacing of 552 μs. To investigate the effect of a restricted FOV, these experiments were repeated with a glass sphere phantom  containing approximately 2 M [1‐13C]lactate and a 2.4 × 2.4‐cm2 FOV. The receiver bandwidth in this case was reduced to 100 kHz , and the echo spacing was therefore extended to 712 μs. To investigate the effect of multiple signal sources, a dual phantom, containing the 7‐mm‐ and 17.2‐mm‐diameter phantoms, was imaged using a 4 × 4‐cm2 FOV.Images were acquired from a cylindrical glass phantom  containing approximately 5 M Pyruvate was hyperpolarized as described previously,13C]pyruvate. Animal experiments were performed in compliance with a project licence issued under the Animals (Scientific Procedures) Act of 1986. Protocols were approved by the Cancer Research UK, Cambridge Institute Animal Welfare and Ethical Review Body.Subcutaneous EL4 lymphomas in C57BL/6J mice13C images covered a 4 × 4‐cm2 FOV, with a receiver bandwidth of 125 kHz, and were from a 6‐mm‐thick slice. The resulting echo spacing was 552 μs. A constant 15° flip angle SpSp pulse was applied alternately to the [1‐13C]pyruvate and lactate resonances, with 1 s between, giving a TR of 2 s for each metabolite. The 11th pair of acquisitions was used as 13C reference scans. T2‐weighted proton images were acquired using an FSE sequence  at the same slice position.The 2.4in vivo were reconstructed in five different ways: using a 13C reference scan13C echoes1H reference scanx direction), which minimized signal in the ghost‐containing background; and using a 2D search .x–ky hybrid space using Ahn's algorithm,k‐space lines were zero‐filled for each partial k‐space acquisition prior to Fourier transformation.Images acquired from phantoms and 13C]lactate image , and the calculated coefficients, α and β, could then be applied to all the k‐space lines in all the images in that series. For the phantom studies, α and β were calculated for each individual image, where the proton image was used to identify regions that contained signal. Where there were multiple signal sources, a single region of interest (ROI) was drawn to include all sources of signal. Two bands, at both ends of the phase‐encoding direction . This area was then moved by half a FOV in the phase‐encoding direction to define areas 1 and 2, and by half a FOV in the frequency‐encoding direction to define areas 3 and 4. For phantom images with a restricted FOV and multiple signal sources, all five areas were drawn manually.Mean values were taken from the magnitude signal. Areas 1/2/3/4/5 are indicated in Figure 313C phantoms are shown in 13C]pyruvate and [1‐13C]lactate images acquired after injection of hyperpolarized [1‐13C]pyruvate into a tumour‐bearing mouse are shown in Figure Images of the 13C reference scan and integrated 13C reference echoes, with a large FOV, a restricted FOV and with multiple signal sources  yielded results similar to those using a search in the [1‐13C]lactate image with the highest SNR, whereas a search in an early‐stage [1‐13C]pyruvate image (second image) led to considerable residual ghosting , 69 images in total, 23 images from each of three mice] and 7.2 ± 5.0%  residual ghosting in [1‐13C]lactate and [1‐13C]pyruvate images, respectively. Similarly, an exhaustive search in the sum of the [1‐13C]lactate images gave 6.0 ± 2.5% (n = 69) and 8.1 ± 5.0% (n = 30) residual ghosting in the [1‐13C]lactate and [1‐13C]pyruvate images.Phase correction using an exhaustive search in a late‐stage [1‐g Figure . When thin vivo dataset. The coefficients obtained were then applied to the lactate image with the highest SNR. Residual ghosting in this latter image and the coefficients obtained from the individual images were then plotted against the SNR of the individual images  for [1‐13C]pyruvate and 15.3 ± 8.7% (n = 3) for [1‐13C]lactate.The 1D search method would have had a clear SNR benefit 413C echo planar images acquired from phantoms and in vivo, showing similar removal of ghosting as the direct 13C reference scan method  and 11.4 ± 5.9% (n = 30) for the 1D and 2D searches, respectively, and, for the in vivo [1‐13C]lactate images, were 6.0 ± 2.5% (n = 69) and 5.6 ± 3.0% (n = 69), respectively. However, noise levels were higher in images corrected using the 2D search because of matrix inversion in the phase correction algorithm. For a modern magnetic resonance scanner, where the gradient coils are well shielded and cross‐term eddy currents are minimized, correction in both the readout and phase‐encoding directions is not required.k‐space displacement in both the kx and ky directions. However, even in this situation, the 1D search method should perform better than the abovementioned reference scan methods as it directly minimizes ghosting in the image domain.A 2D search offered no advantage over a 1D search. Residual ghosting levels in 3.4% n =  and 11.44.21H and 13C signals. Eddy currents, a major source of Nyquist ghosting, are spatially dependent, and a mismatch in location may render the proton reference scan less than optimal for phase correction . For example, a 250‐kHz bandwidth in 13C imaging will require a 1‐MHz receiver bandwidth in the 1H reference scan. Moreover, if the same slice selection gradients are used in acquisition of the 1H reference, the slice thickness will also be one‐quarter of that in the 13C image. A smaller (one‐quarter) slice selection gradient could be used, but then the eddy currents would be different. As these eddy currents may interfere with the eddy currents generated by the readout gradients, different phase errors may be measured in the 1H reference than are actually present in the 13C image.The 1D search is based on a linear phase error model that is similar to that of the widely used reference scan method, which has been proven to be robust on modern clinical systems. The 1D search renders a reference proton scan13C reference echoes13C signals and requires no operator intervention. However, it sacrifices image SNR by prolonging TE. The method proposed here has therefore a substantial SNR benefit in vivo over the integrated reference echoes method. Although clinical systems usually have lower field strengths than the 7 T used here, and therefore longer T2*, this SNR benefit may still hold in clinical applications because of the slower gradient slew rates and the potential need for a larger acquisition matrix to cover a larger FOV. The integrated reference echoes may be inserted at the end of the acquisition train so that the SNR of the image would not be sacrificed; however, this may result in very low signal from the reference echoes.An EPI sequence with integrated 13C phantom. However, a phantom further complicates the spectrum and places more restrictions on SpSp pulse design. Using an image from the phantom for phase correction will also potentially suffer from the same problems as using a proton reference image, where differences in spatial location and eddy currents could lead to suboptimal calculation of the phase correction coefficients.Reference data could be acquired from a 13C imaging because the 13C signals are usually localized. This was the case here, where labelled lactate was observed predominantly in the tumour, but is also the case for other hyperpolarized 13C‐labelled imaging agents, such as the perfusion markers 13C‐labelled urea13C]2‐methylpropan‐2‐ol (t‐butanol).13C]pyruvate images yielded similar phase correction coefficients to those determined from the [1‐13C]lactate image with the highest SNR.The exhaustive search method has not been widely used in the clinic for proton MRI, mainly because many imaging objects cover most of the FOV and, if there is a large overlap between object and ghosting, the exhaustive search method can sometimes lead to heavily biased results. This is not the case for 4.313C reference scan between FOVs of 11.5 and 20 mm (SSIM > 0.98) . Fortunately, the relatively small 13C gyromagnetic ratio and small acquisition matrix (hence high bandwidth in the phase‐encoding direction) make it less prone to off‐resonance effects. Ramp sampling can also help by further increasing the phase‐encoding bandwidth. For example, the 552‐μs echo spacing used here results in an immunity to a B0 shift of ±57 Hz, which can be increased to approximately ±100 Hz when ramp sampling is used with the same gradient amplitude and slew rate. In comparison, the 13C linewidth of [1‐13C]lactate in vivo is usually around 40 Hz on our system. Dynamic central frequency measurements could be incorporated into the experiment by using a simple pulse‐acquire sequence with a small flip angle, although, in our experience, this is unnecessary as the central frequency does not vary much  after the injection of the hyperpolarized medium.Gross shifts and distortions in the 4.4kx–ky–z hybrid k‐space (the hybrid k‐space is obtained by Fourier transformation of the 3D k‐space in the kz direction). The method is also compatible with multi‐channel receive coils. Provided that accelerated acquisition is not used, the search need only be on data from a single channel. When there are strong eddy currents in y or z, a search may be needed in the image from each channel. In the case of parallel imaging, the phase correction coefficients in each iteration of the search should be implemented in the sensitivity map in a similar way to that described by Chen et al.The proposed workflow could readily be applied to 3D EPI, where the 1D search could be performed per slice and the correction coefficients obtained applied to each 513C‐labelled metabolites. This workflow is as capable of removing Nyquist ghosting as a 13C reference scan, at no cost of extra data acquisition during dynamic imaging, and would be straightforward to implement in clinical imaging.We have developed a referenceless EPI workflow for hyperpolarized


With the development of heterogeneous polymer-supported organoselenium catalysts, the solid state 77Se NMR comes to the spotlight. It is necessary to figure out an advanced protocol that provides good quality spectra within limited time because solid state 77Se NMR measurements are always time consuming due to the long relaxation time and the relatively low sensitivity. Studies on small molecules and several novel polymer-supported organoselenium materials in this article showed that cross polarization (CP) method with the assistance of magic angle spinning (MAS) was more efficient to get high quality spectra than the methods by using single pulse (SP) or high power 1H decoupling (HPHD) combined with MAS. These results lead to a good understanding of the effect of the molecular structure, the heteronuclear coupling, the long-range ordering of the solid , and the symmetry of 77Se on quality of their spectra.Characterization of selenium states by  Recently, the catalytic activities of organoselenium compounds have attracted much attention34. Because selenium is a metabolizable element that will not accumulate in the body35, organoselenium catalysts are much safer for both organisms and the environment than transition metal catalysts. Organoselenium-catalyzed reactions usually employed hydrogen peroxide as the clean oxidant that generated no wastes other than the water34. Organoselenium catalysts were very stable and could be recycled and reused for many times without deactivation34. Binding onto polymers, the heterogeneous organoselenium catalysts were even easier to be separated from the reaction solutions and were of profound industrial application values25. During our continuous investigations on organoselenium catalysis34, we found that the selectivity of organoselenium-catalyzed reactions was tunable and different organoselenium catalysts might lead to different reaction paths28. Mechanism studies were very important to understand the protean reaction selectivity.Selenium is a rare element first discovered by Swedish chemist Berzelius in 1818. It has been incorporated in nearly all areas of chemistry now because of the unique chemical and biological properties77Se NMR tests provided direct information of selenium states in reactions and were very efficient tools for mechanism study27. It has a wide chemical shift over 6000 ppm, which is beneficial for producing sufficient resolution to separate signals from different chemical sites37. However, 77Se has a low natural abundance (ca. 7.63%) and low relative NMR sensitivity (6.93 × 10−3)38. The situation gets worse for solid-state NMR (SSNMR) especially when the Se content is even low and more complicated factors are involved. It is well known that the line width in SSNMR spectrum is usually wider than that in solution NMR because of the strong chemical shift anisotropy (CSA), dipolar-dipolar coupling, etc. Magic-angle spinning (MAS) is used routinely in the majority of SSNMR to remove the effects of CSA, to assist in the removal of heteronuclear dipolar-coupling effects, and to narrow lines from quadrupolar nuclei. Traditional single pulse (SP) sequence is always applied with the help of MAS, but still leads to low signal-noise ratio (SNR) for the characterization of 77Se.Besides control experiments,13C, 31P etc43. The method offers a beneficial solution to achieve higher SNR based on the applications of polarization transfer from the abundant proton (1H) spin to low abundance nuclei i.e. 77Se in the present paper via heteronuclear dipole-dipole interactions. The improvements are originated from enhanced sensitivity of the Se and faster longitudinal relaxation time (T1) of 1H compared to 77Se leading to shorter recycle delay time (D1) between every acquisition (≥5T1)46. Experiments through traditional method of SP and a single pulse sequence with special high power 1H decoupling (HPHD) were also performed for comparison with the CP method. The CP method has been successfully applied to determine the state of the low-content selenium in several novel polymers. The novelty of the present paper is that the correlations of intensity of 77Se NMR signal with the solid state and molecular structure of polymers were demonstrated for the first time. Herein, we wish to report our findings.The aim of the present article is to find out factors that affect the solid NMR spectrum and the best protocol to get high quality spectrum for several novel selenium-contained polymers. A method by using a combination of cross-polarization (CP) and MAS was applied, which has been previously used for the characterization of 77Se. PXRD pattern of H2SeO31 is completely different from that of PhSeO2H 2, confirming their different crystal structures due to the presence of a benzene ring47. In contrast, all the polymer materials exhibit a broad reflection, indicating their amorphous structures48.Solid-state properties of the samples were initially investigated. Figure 2SeO31 was first measured and used as a secondary external reference standard. Figure 77Se NMR spectra obtained by the different pulse sequencesas described in experimental section: single pulse (SP), high power 1H decoupling (HPHD) and cross polarization (CP). To well illustrate the efficiency of the different methods, the intensity of the isotropic center-bands was normalized. A significant spinning side band manifold separated by the rotation frequency as a result of the chemical shift anisotropy (CSA) was observed in Fig. 2SeO3 crystal49. The chemical shift obtained by SP sequence was chosen to be the reference standard (1288.1 ppm). Full Width at Half Maxima (FWHM) and the SNR of the isotropic band were obtained and listed in Table 1H would broaden the line of 77Se spectra due to heteronuclear dipolar coupling between 1H and 77Se, which could be removed by 1H decoupling. Hence, the decoupling could improve SNR significantly and reduce FWHM, which explained the improvement for both HPHD and CP because of both of them including a decoupling pulse sequence. Moreover, the improvement for the CP was also related to the efficiency of polarization transfer between 1H and 77Se. It should be mentioned that CP needed less time to achieve comparable results as HPHD due to the shorter D1 , and its chemical shift was 1127.4 ppm with respect to H2SeO3  to 45 (HPHD) and to 35 (CP), and the FWHM was reduced from 109 Hz (SP) to 82 Hz (HPHD) and to 85 Hz  was determined to be 1.46 mmol/g by an acid-base titration with NaOH. If all the benzenes were selenized, the Se content should be 4.33 mmol/g. Hence, only around 1/3 of benzenes in the polymer were substituted (Scheme 1). Accordingly, similar low-content of Se could be found in polymer 4 (-Se-CH3), which lead to low sensitivity of 77Se in NMR measurement. Figure 4 obtained by the three methods. Several strong and wide spinning side bands could be observed in Fig. 37. In contrast to compounds 1 and 2  was measured by SSNMR with the same experimental parameters applied for polymer 4. Both 79Br and 81Br are half-integer (I = 3/2) spins with large quadrupole moments , which were so large that yieldedextensive line broadening53. As a result, the sensitivity of Se in this polymer was too weak to give a reliable result under the same conditions as polymer 4 (Data not shown). As for the polymer 6 , the width of the isotropic bands became narrow , and correspondingly improved the SNR values from 11 to 21  could be observed in the spectrum of polymer 6 (b), indicating fully transformation from polymer 4 to polymer 6, which manifested that SSNMR could characterize the Se state in polymers even under low content.A comparison with the same spectrum range for polymer The Se state in amorphous polymers even under low content was able to be efficiently determined by SSNMR through CP methods. It was found that low symmetry of molecular structure and less long range ordering structures of the solid would broaden the signal and enlarge the spinning side bands and conseqeuntly reduce SNR. These results would provide preliminary information for further investigation about the characterization of solidified selenium-containing materials from both molecular level perspective and macrostructure perspective.1 (H2SeO3) and benzeneseleninic acid 2 (PhSeO2H) were commercially available. The selenium-containing polymers 4–6 were prepared from polystyrene was first immersed in cyclohexene (30 mL) overnight under N2. 5.6 mL of tetramethylethylenediamine  and 48 mmol of n-BuLi  were added. The mixture was stirred at 65 °C for 4 h and filtrated. The polymer was then washed by 10 mL of THF for three times and removed into a flask by washing with 40 mL of THF and mixed with 5 mmol of (MeSe)2 at 0 °C. After stirring 20 min at 0 °C, water (3 mL) was added and the color of the polymer turned yellow. The polymer was washed with THF , H2O , methanol , CH2Cl2  and ether  subsequently and dried under vacuum at 65 °C for 24 h to provide the polymer 4.4.0 g of polystyrene resin 4 was immersed in chloroform (60 mL) under N2 overnight. A solution of 6 mmol of Br2 in CHCl3 (20 mL) was added and stirred for 30 min cooled with ice-water.The resin was then washed with anhydrous EtOH and removed into a flask containing 60 mL of anhydrous EtOH. The color turned red after heating at 70 °C for 2 h and after filtration, the polymer was washed with EtOH  and CH2Cl2  subsequently and then dried at 65 °C under vacuum for 24 h to produce the polymer 5 (4.3 g).4.0 g of polymer 5 was immersed in 60 mL CHCl3 under N2 overnight. 10 mmol of H2O2 (30w/w %) was added at 0 °C. The color of the polymer turned white gradually after stirring at 25 °C for 2 h. After filtration, the resin was washed with EtOH  and CH2Cl2  subsequently and then dried at 65 °C under vacuum to produce the pure polymer 6 (0.6849 g). Acid–base titration with NaOH indicated that the content of Se in polymer 6 was 1.46 mmol/g.0.71 g of polymer 77Se NMR was performed on Bruker Avance III spectrometers operating magnetic field strengths of 9.4 T, corresponding to Larmor frequencies at 76.3 MHz for 77Se. A Bruker 4 mm double resonance HX MAS probe was used. H2SeO3 was used as a secondary external reference standard for PhSeO2H and polymers, and its isotropic chemical shift (δ) was calibrated to be 1288.1 ppm with respect to dimethylselenide (0.0 ppm). Three pulse sequences were applied , and the composite pulse 1H decoupling program is spinal64.The decoupling power was set to be 66.72 W with the pulse length of 9.0 μs. 3, cross polarization (CP) transfer from 1H spin using optimized contact pulse durations of 4.5 ms or 5.0 ms (ramped for 1H), and two-pulse phase modulation (TPPM) 1H decoupling during acquisition . The decoupling power was set to be 66.72 W with the pulse length of 8.4 μs. 90° radio frequency (rf) pulse length for1H excitation was 4.2 μs. In order to shorten recycle delay time (D1) between each acquisition, a 30°rf pulse of 1.54 μs  was applied instead of the normally used 90° rf  for 77Se excitation in SP and HPHD pulse sequences. The applied D1 are listed in Table 2SeO3 and PhSeO2H are 12~15 min and 10~15 h, respectively, and the duration for the polymers is 24 h.Solid-state


Instability of the knee, related to anterior cruciate ligament injury, is treated by surgical reconstruction. During recovery, a loss of proprioceptive input can have a significant impact. Few studies have evaluated the benefits of rehabilitation of the knee in aquatic environment on functional outcomes.This study aimed to compare an innovative rehabilitation protocol combining reduced conventional rehabilitation with aquatic rehabilitation, with a conventional rehabilitation, according to the National French Health Authority, in terms of kinetics, development of proprioceptive skills, and functional improvement of the knee.67 patients, who were amateur or professional athletes, were randomized into two groups: 35 patients followed the conventional rehabilitation protocol (Gr1) and 32 patients followed the innovative rehabilitation protocol (Gr2). Patients were evaluated before surgery, and at 2 weeks, 1, 2, and 6 months after surgery using posturography, and evaluation of muscular strength, walking performance and proprioception. This study is multicenter, prospective, randomized, and controlled with a group of patients following conventional rehabilitation (level of evidence I).For the same quality of postural control, Gr2 relied more on somesthesia than Gr1 at 6 months. The affected side had an impact on postural control and in particular on the preoperative lateralization, at 2 weeks and at 1 month. Lateralization depended on the affected knee, with less important lateralization in Gr2 preoperatively and at 1 month. The quadriceps muscular strength was higher in Gr2 than in Gr1 at 2 and 6 months and muscle strength of the external hamstring was greater in Gr2 than in Gr1 at 6 months. The isokinetic test showed a greater quadriceps muscular strength in Gr2. Gr2 showed a greater walking distance than Gr1 at one month. Gr2 showed an improvement in the proprioceptive capacities of the operated limb in flexion for the first 2 months.The effectiveness of the innovative rehabilitation program permits faster recovery, allowing for an earlier return to social, sporting, and professional activities. Faster retrieval of knee function following aquatic rehabilitation would prevent both short-term risk of lesions of the contralateral limb due to overcompensation and long-term risk of surgery due to osteoarthritis.NCT02225613. Anterior cruciate ligament (ACL) injury is one of the main injuries in sporting activities with an incidence of nearly 32,000 victims per year in France , especiaThe main objective of this study was to compare an innovative rehabilitation protocol with an “aquatic part”  and a “dry part” and a conventional rehabilitation protocol  in termsSixty-seven patients aged from 18 to 49 years participated in this study. The volunteers underwent surgery by orthopedic surgeons from the University Hospital of Nancy and private clinics around Nancy, East of France. Among the 67 patients, none have undergone surgical complications.All subjects were leisure or competitive sportsmen . All of them had chronic knee instability and the indication of a first-line ACL reconstruction using TLS, semitendinosus or gracilis tendons or KJ was given. Exclusion criteria were history of neurological disease , medication for psychotropic or antihypertensive purposes, contraindications to aquatic activities , and recent sprains less than 3 months in the lower limbs that could interfere with postural control.ClinicalTrials.gov with the identifier: NCT02225613.This study is multicenter, prospective, randomized, and controlled with a group of patients following conventional rehabilitation (level of evidence I). All participants gave written informed consent before the randomization. The study was approved by the French Medical Ethical Committee (Comité de Protection des Personnes de Lorraine), realized in structures  agreed for research , and registered in n = 35; mean age = 29.91 ± 7.70 years; 21 males) followed a conventional rehabilitation protocol (according to HAS recommendations) and the second group  followed an innovative rehabilitation protocol with a reduced conventional part and an aquatic part. The total duration of patient care was the same between the two groups regardless of the rehabilitation protocol followed: 45 min, once a day, five times a week, from Monday to Friday, for 3 weeks. After these 3 weeks, all patients returned to a conventional rehabilitation (according to HAS recommendations) , until tissues healing . For 3 wPostural control tests, main endpoint, were carried out in a specially designed sound proof room devoted to posturography recordings. A vertical force platform, fitted with three strain-gauge force transducers  was used to perform posturography and to provide a measurement of the body sway in terms of displacement of the center of foot pressure (CoP) in a two-dimensional horizontal plane  Figure . The meaSOM), ratio C4/C1, the visual contribution (RVIS) and ratio C5/C1, the vestibular contribution (RVEST)  was calculated by adding the scores of each condition, and dividing that sum by six [MES = sum C1–C6)/6], low values being representative of good postural control –22. Each (RVEST) , 24 , and four times after the beginning of rehabilitation; at 2 weeks (Ev2) (corresponding to the beginning of the specific rehabilitation to the study), at 1 month Ev3) (corresponding to the end of the specific rehabilitation to the study), at 2 months (Ev4) and at 6 months (Ev5) The intensity of affected knee joint pain was assessed with the Visual Analog Scale (VAS) for pain. The VAS consisted of a 100 mm line whose endpoints were designated as “no pain” (at 0 mm) and “unbearable pain” (at 100 mm), respectively. Patients were requested to locate the level of knee pain on the line with a small vertical mark , 26.(2)The proprioception test was carried out in a sitting position, on an examination table, without contact with the base of the lower half of the posterior part of the thigh. The tested member was placed on a skateboard. Flexion and extension movements were realized with the patients’ eyes closed. The lower limb was passively flexed or extended by the physiotherapist at an arbitrary angle before being returned passively to the neutral position. Patients were instructed to flex actively the tested limb to the same angle than the passive position determined by the physiotherapist –31. The (3)Joint amplitudes were evaluated in flexion and extension, active and passive , 32, 33.(4)Trophicity refers to all the mechanisms and processes involved in the nutrition of organs and tissues. This test consisted of digital pressure on the edema after which it was observed whether the imprint left by the finger disappeared rapidly or not. This test was positive if the impression disappeared slowly .(5)−1 and then 180°s−1.Muscle strength testing was performed in knee extension to evaluate the strength of the femoral quadriceps, and then in knee flexion to assess strength of the hamstrings. A score ranging from 0 (no contraction) to 5  was attributed to muscle strength . An isok(6)The 6 min walk test consisted of measuring the greatest possible distance that a patient could walk at their own pace in 6 min .(7)Patients had to respond to the Lysholm-Tegner , InternaThe functional improvement, secondary endpoints, was evaluated by different clinical assessments:For each of the five evaluation times, the clinical tests performed are presented in Table n) and percentage (%). Quantitative data were expressed as mean and SD.Qualitative data were expressed in terms of number  version 23.0 was used for all analyzes.p = 0.427).No statistically significant difference was observed for anthropometric variables  between the two groups of patients at baseline Table . No signVIS and RVEST were observed between the two rehabilitation groups at the five evaluations whereas RSOM score was significantly lower in Gr2 than in Gr1 (p = 0.008) at Ev5, reflecting a greater use of somesthesia in balance control (Table p = 0.002), Ev2 (p < 0.001) and Ev3 (p = 0.001). Gr1 rely more than Gr2 on the contralateral limb to the surgery at Ev1 (p = 0.037) and Ev3 (p = 0.039).No statistically significant differences of the MES of the sway path traveled and the area covered by the center of foot pressure were observed between the two rehabilitation groups at the five evaluations. No statistically significant differences of Rol Table . PatientVIS and RVEST in both groups. Patients used more visual and the vestibular input at Ev2 to maintain their balance control . In Gr1, the area decreased between Ev2 and Ev3 (p = 0.004) and between Ev3 and Ev4 (p = 0.001), and the sway path decreased between Ev3 and Ev4 (p = 0.001). In Gr2, the sway path increased between Ev1 and Ev2 (p = 0.003) and decreased between Ev2 and Ev3 (p = 0.001). Gr1 relied more on the limb contralateral to the operated limb between Ev1 and Ev2 (p < 0.001), whereas they relied less on the contralateral limb between Ev2 and Ev3 (p < 0.001) and Ev1 and Ev5 (p = 0.010). Gr2 relied more on the limb contralateral to the surgery between Ev1 and Ev2 (p < 0.001), whereas they relied less on the contralateral limb between Ev2 and Ev3 (p < 0.001).Significant differences were showed between Ev1 and Ev2 for R(1)p = 0.007) and Ev1 and Ev5 (p = 0.039). In Gr2, pain increased between Ev1 and Ev2 (p = 0.027), then decreased between Ev2 and Ev3 (p < 0.001), Ev4 and Ev5 (p = 0.030), and Ev1 and Ev5 (p = 0.012).No statistically significant difference in pain was observed between the two rehabilitation groups over time. In Gr1, pain decreased between Ev2 and Ev3 ((2)p = 0.002).No statistically significant difference in proprioception was observed between the two groups over time. In Gr2, an improvement of proprioception in flexion was observed at Ev4 ((3)p < 0.001), Ev3 and Ev4 (p < 0.001), Ev4 and Ev5 (p < 0.001). In Gr1, an improvement in passive extension was observed between Ev2 and Ev3 (p = 0.015) and Ev3 and Ev4 (p = 0.006), and an improvement in active extension was observed between Ev4 and Ev5 (p < 0.001). In Gr2, an improvement in active extension was observed between Ev2 and Ev3 (p = 0.008), Ev3 and Ev4 (p = 0.038), and Ev4 and Ev5 (p = 0.002). An improvement in passive extension was observed between Ev2 and Ev3 (p = 0.021) and Ev4 and Ev5 (p = 0.006) in Gr2.No statistically significant difference in joint amplitude was observed between the two groups over time. In the two groups, an improvement in active and passive flexion was observed between Ev2 and Ev3 ((4)p = 0.039; Gr2: p = 0.006) and Ev4 and Ev5 .No statistically significant difference in trophicity was observed between the two groups over time. An improvement in trophicity was observed in the two groups between Ev2 and Ev3 (Gr1: (5)p = 0.015) and Ev5 (p = 0.027) between the two groups. At Ev4 in Gr2, 14 patients obtained a score of 5 (43.75%), 17 a score of 4 (53.13%) and 1 a score of 3 (3.12%) versus 5 a score of 5 (14.29%), 25 a score of 4 (71.43%) and 1 a score of 3 (2.85%) in Gr1. At Ev5 in Gr2, 24 patients obtained a score of 5 (75%) and 8 a score of 4 (25%) versus 17 a score of 5 (48.57%) and 18 a score of 4 (51.43%) in Gr1. Statistically significant differences in internal hamstring muscle strength were observed at Ev2 (p = 0.026). In Gr2, 13 patients obtained a score of 4 (40.63%), 14 a score of 3 (43.75%), and 5 a score between 0 and 2 (15.62%) versus 8 a score of 4 (22.86%), 26 a score of 3 (74.29%) and 1 a score between 0 and 2 (2.85%) in Gr1. Statistically significant differences in external hamstring muscle strength were observed at Ev5 (p = 0.019) between the two groups. In Gr2, 22 patients obtained a score of 5 (68.75%), 9 a score of 4 (28.13%) and 1 a score of 3 (3.12%) versus 12 a score of 5 (34.29%), 21 a score of 4 (60.00%) and 2 a score of 3 (5.71%) in Gr1. The statistical analysis showed an increase in the muscle strength of the quadriceps between Ev2 and Ev3 (p = 0.006) and Ev3 and Ev4 (p = 0.039) in Gr1. The muscle strength of the hamstring increased between Ev2 and Ev3 (p = 0.001) and Ev4 and Ev5 (p = 0.031). In Gr2, the muscle strength of the quadriceps increased between Ev2 and Ev3 (p < 0.001), and the muscle strength of the hamstring increased between Ev2 and Ev3 (p < 0.001). At Ev5, the muscle strength of the quadriceps developed in isokinetic testing was significantly greater in Gr2 at either 60°s−1 (p = 0.027) or 180°s−1 (p = 0.047). Moreover, muscle deficit of the quadriceps of the ipsilateral limb compared to the contralateral limb was lower in Gr2 (p = 0.020).Statistically significant differences in quadriceps muscle strength were observed at Ev4 ((6)p = 0.025). Gr1 traveled 425.27 ± 158.45 m against 506.59 ± 127.70 m for the Gr2. 6 min walking performance significantly increased at Ev5 compared to Ev3 in the two groups (p < 0.001) and there was no significant difference at Ev5.A statistically significant difference in the 6 min walk test was observed between the two groups at Ev3 ((7)p < 0.001) and Gr2 (p = 0.004). Statistical analysis showed an improvement in the IKDC functional score in both groups (p < 0.001). Significant improvement in knee function after injury was observed in both groups (p < 0.001). In both groups an improvement was observed for pain , daily life , quality of life  and an increase in sports and leisure activities .No statistically significant difference for questionnaires was observed between the two groups. An improvement in the functional Lysholm-Tegner score was observed between Ev1 and Ev5 in Gr1  improves proprioception and limits overcompensation on the limb contralateral to the operated limb. Even if patients undergoing a conventional rehabilitation protocol recover the delay after 6 months of surgery, faster and better recovery of knee functionality, following aquatic rehabilitation would in the short-term prevent injury to the contralateral limb as a result of overcompensation, and serve in the longer term to reduce the risk of osteoarthritis. The effectiveness of such rehabilitation could also enable patients to recover social, physical and professional activities earlier, which would also be of economic benefit, in particular with a reduction in work absence.ClinicalTrials.gov with the identifier: NCT02225613.All participants gave written informed consent before the study, which was approved by the French Medical Ethical Committee (Comité de Protection des Personnes de Lorraine) and registered in All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The complexation of molecular clips containing fragments of diphenylglycoluril and benzocrown ethers with paraquat and its derivatives has been studied both in solution and in the solid state. In this paper we studied the influence of the crown ether ring size and the nature of the substituents at the nitrogen atoms of the paraquat derivatives on the composition and stability of these complexes. Chen anetone-d6 . Recentletone-d6 .1–5, and studied their complexation with alkali metal [6 with paraquat (7) and its analogues 8–10  and its derivatives are among the most studied electron-acceptor guests that can form stable inclusion complexes with electron-donating molecules. Stabilization of such inclusion complexes is mainly realized due to π–π stacking and C–H∙∙∙X  interactions. The stability of inclusion complexes based on molecular clips and paraquat derivatives depends on two factors: 1) the value of the positive charge on the dipyridinium fragment of paraquat derivatives, the distribution of which is well described by the molecular electrostatic potential  the 1–6, we used the approach approved earlier and described in our previous studies [For qualitative estimation of the complexing properties of the obtained molecular clips  studies –30.FAB–MS is known as a versatile method to detect supramolecular complexes from solutions after transferring them into the gas phase –32.1–5 and paraquat (7) in 3-nitrobenzyl alcohol were subjected to mass spectral analysis. Ion peaks resulting from the loss of the hexafluorophosphate anion from 1:1 complexes of the appropriate molecular clip with paraquat (7) have been observed in the mass spectra , significant upfield shifts of Нβ aromatic proton signals of paraquat  have been observed as a result of their shielding by aromatic fragments of clip  as well as signals of methyl and methylene protons of the guests have been revealed  appears. The new band was assigned to a charge-transfer complex formed by the π-donor aromatic fragments of clips 1–5 and the π-acceptor dipyridinium fragment of paraquat derivatives 7–10 located in the pseudo cavity of the clip [1–5 constant and systematically varying the concentration of paraquat derivatives 7–10. The obtained experimental data were processed by the non-linear method of least squares using the SIRKO program [2 with paraquat (7) are presented in The analysis of UV–vis spectra could also provide qualitative information on the formation of inclusion complexes in solution. The addition of guests the clip . The int program . The val1–3 are best described by titration curves corresponding to 1:1, and for clips 4 and 5 – to 1:1 and 1:2 complexes. Stability constants of the complexes are presented in The data obtained for molecular clips 7) is increased by ≈0.6 kcal/mol with increasing of the polyether macrocycle size by one oxyethylene fragment reaching a maximum for the clip 4 (6 with paraquat (7), which was: lgK = 1.46 ± 0.01 . The complex of clip 6 with paraquat (7) may be stabilized through π–π stacking interactions of the electron-deficient aromatic rings of paraquat and the electron donating veratrol fragments of the clips, as well as through hydrogen bonds between the Hα, Hβ, and CH3 protons of paraquat with oxygen atoms of carbonyl groups in glycoluril fragment.On the average, the stability of clip complexes with paraquat , whereas the polyether chain – to that of the complexes of the clips 3–5 with paraquat (7). The data obtained show that the clips 4 and 5 are maximum pre-organized to form a 1:1 complex with paraquat compared to other clips with benzocrown ether moieties.Therefore, the glycoluril fragment makes the predominant contribution to the complex stability of clips 2, 3 and 5 with paraquat (7) have been studied by X-ray crystallography. All these clips form inclusion complexes with a 1:1 ratio where the paraquat molecule is located within the pseudo-cavity formed by two crown ether fragments . Additionally the host–guest structures are stabilized by weak C–H···π interactions of the hydrogen atoms of the methylene groups and dipyridinium fragments of paraquat (the corresponding H···π distances are ≈2.84 Å).The complexes are stabilized by π–π intermolecular stacking interactions between the electron-donating aromatic fragments of the clips and the electron-deficient dipyridinium fragments of paraquat. The distances between the centroids of these fragments are 4.07 Å for In the crystal phase molecules form infinite rows in such way that the neighboring molecules are turned to each other on 180° within the row .Within the row molecules are arranged by π–π stacking interactions between the side aromatic fragments of the clips with a centroid–centroid distance in the range of 3.51–3.71 Å. There are acetonitrile solvate molecules and hexafluorophosphate anions between neighboring rows.8–10 with molecular clips 1–5 by weakening of π–π stacking and C–H···O interactions [8–10 may increase the stability of complexes by forming additional hydrogen bonds between molecular clips and hydroxy groups of the substituted dipyridinium salts [Quantum chemical calculations show that the substitution of methyl groups in the molecule of paraquat by oxyethylene linker results in a decrease of the positive charge on the dipyridinium fragment  that, inractions . At the um salts –40.1–5 and paraquat derivatives 7–10 in acetonitrile are shown in 1–5 with paraquat derivatives 7–10 stability constants K2 have been found to vary in the range of 7–10 except for 3@10 with K2 equal to 49. Substitution of the methyl group in the 7 by ethylene or diethylene glycol fragments leads to an increase in stability of the complexes except 5@8 and 5@9 (1–3 with paraquat derivative 10 are similar compared to complexes with paraquat (7) (1@10) or slightly lower .The stability constants of clips  and 5@9 . The com8 and 9, it would be logical to assume that the increase of the complex stability is caused by the formation of additional hydrogen bonds involving the substituents on the nitrogen atoms, including terminal hydroxy groups. The fall in complex stability of clips 1–3 with 10 probably occurs due to the fact that longer oxyethylene chains form less energetically favorable hydrogen bonds. The experimental data obtained on the stability of molecular clips 1–5 with paraquat (7) in solution is well correlated with the data for these complexes in the crystalline state revealed by quantum chemical calculations [10 , the host–guest complexes 2@8 and 3@8 are stabilized by π–π stacking, C–H···O and C–H···π interactions. So far as the structures of these complexes are very similar the main attention will be paid to some differences only. The presence of the terminal hydroxy groups creates the capability of additional stabilization of the complexes of the clips with paraquat derivatives due to the formation of the hydrogen bonds. Indeed, such an hydrogen bond is observed between one hydroxy group of cation 82+ and the polyether oxygen atom of clip 3 . But a similar hydrogen bond is absent in the complex 2@8. One hydrogen bond is formed by the hydroxy group of 9 and the carbonyl oxygen atom of the diphenylglycoluril fragment of the clip  in the complexes 2@9 and 3@9. The other hydroxy group of 9 participates in the formation of the hydrogen bond with the polyether oxygen atom of clip 2 within this complex while the intermolecular hydrogen bond between such a hydroxy group and the polyether oxygen atom of the clip belonging to the neighboring complex was found in the crystals of 3@9. In the crystal phase these complexes form the infinite rows that consist of alternate molecules inverted to each other as it was found for the complexes of clips with paraquat (7).Similar to complexes with paraquat . The oxygen atom of the second hydroxy group acts as proton acceptor and forms the weak C–H···О hydrogen bonds with CH2 and Hα hydrogen atoms of 10 belonging to the neighboring complex .For example, the intramolecular hydrogen bonds with participation of the carbonyl oxygen atom which are typical for complexes The crystal packing is similar to the one described above for other complexes. The π–π stacking interactions between the side aromatic fragments have the major contribution in the mutual disposition of studied complexes. A lot of hydrogen bonds have been found between neighboring complexes or between complexes and solvate molecules and counterions.3@10 both crown-ether cavities are occupied by water molecules. Each of these water molecules form two strong hydrogen bonds with polyether oxygen atoms . Moreover, some of the water molecules participate as a proton acceptor in the formation of the C–H···О hydrogen bonds with the methylene hydrogen atoms of the glycoluril fragment of neighboring molecules . The hydroxy groups of 10 participate in the intermolecular interactions with the carbonyl oxygen atoms of neighboring molecules only forming two hydrogen bonds . As a result, the crystal packing of this complex differs from the described above. The molecules within the infinite rows are bonded by the hydrogen bonds rather than π–π stacking interactions.In the case of complex 7) preferably of 1:1 composition. The stability constants with paraquat (7) rise with the increase of the polyether cycle size. The maximum complementarity of the pseudo cavity for binding of paraquat (7) was observed for clips based on benzo-21-crown-7 and benzo-24-crown-8. The introduction of substituents with terminal OH groups on the nitrogen atoms of paraquat usually increases the stability of complexes formed, which is caused by their additional stabilization due to hydrogen bonds between the terminal hydroxy groups and oxygen atoms of the polyether chains, and the carbonyl groups of the molecular clips. In the solid state, the studied clips form inclusion complexes of 1:1 composition.Thus, the data obtained allows us to conclude that molecular clips based on diphenylglycoluril and benzocrown ethers form inclusion complexes with paraquat (File 11H NMR and FAB–MS spectra are provided.Experimental section, complete X-ray data,


A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds. The activity enhancement of the glucose oxide and horseradish peroxidase enzymatic cascade on DNA scaffolds has been linked to proximity effects. Here, the authors challenge this view and suggest that the activity improvement is rather due to the pH near the DNA surface. Enzyme cascades, which sequentially perform multiple enzymatic reactions, have a critical role in signal transduction and amplification in biological systems and also have a wide range of potential applications in biotechnology12346891011et al.et al.1215Glucose oxidase (GOx) and horseradish peroxidase (HRP) are the most frequently used enzyme pair to demonstrate the feasibility and advantages of multienzyme systems on DNA scaffolds. Müller and Niemeyeret al.et al.18131117However, the mechanistic understanding of these experiments is still unsatisfactory. First, although the enzyme pairs were constructed by similar strategies, the activity enhancements vary a lot. For instance, Fu Here we examined the putative proximity effect for the GOx–HRP cascade without relying on DNA nanotechnology. We place GOx and HRP within 2 nm of each other using a crosslinker, show that the catalytic activity of both enzymes is retained and demonstrate that there is no enhancement in cascade throughput (either transiently or permanently) and no direct substrate transfer. As the proximity channeling is ruled out, the DNA scaffold must be responsible for the previously observed activity enhancement. We measure the pH dependence of the maximal reaction rate of GOx and HRP and find that the observed activity enhancement can be explained by the lower pH near the DNA surface where the enzyme is located. These findings challenge the traditional view of proximity channeling and provide new insights into the role of DNA scaffolds.D-glucose with oxygen and produces glucono-δ-lactone and hydrogen peroxide -diammonium salt (ABTS). Consumption of 54 μM oxygen in 1 h corresponded to the formation of 112 μM ABTS+·, proving that the stoichiometry of H2O2 to ABTS+· is 1:2. It also confirmed that the conventional assay using the GOx/HRP cascade is reliable as the high concentration of HRP (20 nM) ensures the rapid and complete conversion of H2O2 produced by 1 nM GOx. The colorimetric assay yielded a rate of H2O2 generation of 15.4±0.1 nM s−1 (mean±s.d.) in 1 mM glucose (the Michaelis–Menten kinetics of glucose is shown in 2O2 to 1 nM HRP and 2 mM ABTS (Km of HRP for H2O2 of 2.55±0.11 μM and a turnover number kcat of 32.7±0.4 s−1 (mean±s.e.). The concentration of the H2O2 stock solution has been determined by measuring the amount of ABTS+·, which can be generated from a given H2O2 solution by HRP . There is a clear transient stage at the beginning in the case of 1 nM GOx with 1 nM HRP . The fact that the final activity converges to an identical value demonstrates that the only potential benefit of an improved transport is a shortening of the initial transient stage if the intrinsic kinetics of the involved enzymes do not change. This conclusion had been drawn in the literature decades ago when biochemists were first defining the concept of substrate channeling222324All of these reactions converge to a state in which the H+· production was modelled with a system of ordinary differential equations using the kinetic parameters measured individually for GOx and HRP (2O2 is well mixed. A detailed justification for this fact is given in our previous work2O2 generated by GOx can diffuse from a GOx molecule to a HRP molecule in 0.6 ms (the diffusion coefficient of H2O2 is 1.71 × 10−9 m2 s−1 at 25 °C (ref. 2O2. As the maximum reaction velocity of the HRP (Vmax is 33 nM s−1 for 1 nM HRP) always exceeds the production of H2O2 by GOx (15 nM s−1 for 1 nM GOx), the H2O2 concentration converges to a constant value so that the actual reaction velocity of HRP equals 15 nM s−1 (The time course of ABTS and HRP . This mo °C ref. ). As it 5 nM s−1 .+· production by half. The H2O2 concentration quickly exceeds the Km of HRP and continues to rise throughout the experiment , whereas improving the activity of the rate-limiting enzyme can significantly and permanently enhance the throughput.The results above demonstrate that the diffusion of the intermediate substrate HN-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), with a spacer length of 8.3 Å. As the free cysteine residues of the GOx are hard to access, we grafted more sulfhydryl groups from the lysine residues via N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) modification and subsequent dithiothreitol thiolation  and found that many encapsulated enzymes showed much higher activities than their free form. Specifically, the kcat for the HRP increased from 32 to 290 s−1 after encapsulation, and other enzymes such as GOx, malate dehydrogenase and lactate dehydrogenase all showed increased activities. This suggests that the DNA itself was the major reason for the increase in the enzymatic activities. Zhao and colleagues suggested that the enhancement is because the hydration layer attracted by negatively charged DNA can stabilize the enzymes located around the highly ordered water molecules. However, the ordered water layer only contributes within a few angstroms33Recently, significant enhancement of throughput for the GOx–HRP cascade was reported in giant DNA structures. Linko ψ by We believe that the many negative charges on large DNA structures, and in particular on DNA origami, are very important for the activity of conjugated enzymes. Similar to polyelectrolyte films37s is the pH at the surface, pHb is the pH in the bulk solution, F is the Faraday constant, R is the universal gas constant, and T is the temperature.where pHet al.13 cm−2, the surface potential is also about −130 mV. The density of negative charges on DNA origami is significantly higher, although a theoretical description of the resulting surface potential is not straightforward−2 , HRP, D-glucose, H2O2 (30 wt.% in water) and ABTS were purchased from Sigma Aldrich Co. LLC, St Louis, MO, USA; SPDP and sulfo-SMCC were purchased from ThermoFisher Scientific, Inc., USA. For all the experiments, the purchased HRP was first purified by size exclusion chromatography to remove the impurities (A403/A280) of purified HRP is 2.5±0.1.GOx from purities . The RZ 5 and 2.61 × 104 M−1 cm−1, respectively. The extinction coefficient for HRP at 403 nm is 1.0 × 105 M−1 cm−1. Enzymatic activities were measured from the changes in absorbance at 415 nm (or 600 nm) by the spectrophotometer .All of the enzyme quantifications were carried out on a ultraviolet–visible spectrophotometer . The extinction coefficients for GOx at 280 and 450 nm are 2.67 × 10For the GOx kinetic assay, 20 μl of 50 nM GOx was added to 980 μl of substrate solution  to initiate the reaction. The final assay solution contains 2 mM ABTS, 1 mM glucose, 1 nM GOx and 20 nM HRP. The increase in absorbance at 415 nm was recorded. The activity was computed from the slope of the absorbance versus time curve during the first 1 min.D-glucose in a sealed vessel. We placed the probe of the dissolved oxygen meter in a glass vessel fully filled with 3 ml substrate buffer. As soon as 10 μl of 300 nM GOx was added to the solution , the vessel is immediately sealed with a piece of plastic paraffin film. The oxygen concentration was recorded for 1 h under magnetic stirring.The consumption of oxygen by GOx was measured by a dissolved oxygen meter  in PBS buffer containing 1 mM 2O2 and 20 μl of 2 mM ABTS solution was added to 940 μl of PBS buffer . The absorbance at 415 nm (or 600 nm) was recorded for the first 2 min.For the HRP activity assay, 20 μl of 50 nM HRP, 20 μl of 10 mM HD-glucose. The absorbance at 600 nm was recorded for 1 h.For the GOx–HRP cascade reaction, the final assay solution contains 1 nM GOx, 1 nM HRP, 2 mM ABTS and 1 mM All the assays were replicated at least twice. We also carried out the experiments with different batches of samples. For the time-course curve, as the zero point drift and the time offset for mixing slightly vary from parallel experiments, we do not average them but analyse them individually.All the assays were carried out at 23±1 °C.The HRP and GOx was conjugated with a bi-functional linker, sulfo-SMCC. As the two free cysteine residues on GOx are hard to access, free sulfhydryl groups were first grafted from the lysine residues on the surface of GOx. In a typical reaction, 40 μl of 80 mM SPDP solution (in dimethyl sulfoxide) was added into 1 ml of 40 μM GOx  with a ratio of 80:1. After stirring for 1 h at room temperature, 20 μl of 1 M dithiothreitol was added to the mixture for 30 min. The excess linker and dithiothreitol were removed by passing the solution twice through a Hitrap desalting column . The concentration of GOx–SH was determined from its absorbance at 280 nm.HRP was functionalized via the maleimide group with sulfo-SMCC. Typically, 80 μl of 40 mM sulfo-SMCC solution (in 50 °C water) was added dropwise into 500 μl of 40 μM HRP solution . The mixture was stirred for 1 h at room temperature. The HRP–SMCC was purified by passing it through a Hitrap desalting column twice. The concentration of HRP–SPDP was determined by its absorbance at 403 nm.For the conjugation of GOx and HRP, GOx–SH solution was added into the purified HRP–SMCC solution at a molar ratio of 1: 1. The mixture was incubated at room temperature for 1 h and stored at 4 °C before separation. The conjugated enzymes were separated by size exclusion chromatography with a HiLoad 16/600 Superdex 200 prep grade column on an ÄKTA system.2O2 (diffusion coefficient: 1.71 × 10−9 m2 s−1) can diffuse the average distance between enzyme pairs (about 1 μm for the system containing 1 nM GOx and 1 nM HRP) in 0.6 ms, the H2O2 solution can be considered well mixed on the timescale of the experiment, and the cascade reaction is determined by the kinetics of GOx and HRP. The production rate of H2O2 depends on the GOx activity  at time t; r1 is the generation rate of H2O2 by GOx, which is approximately constant in our experiment; kcat,1 is the turnover number of GOx; and Vmax,2 and Km,2 are the maximal velocity and Michaelis constant of HRP, respectively.where [Data supporting the findings of this study are available within the article and its How to cite this article: Zhang, Y. et al. Proximity does not contribute to activity enhancement in the glucose oxidase–horseradish peroxidase cascade. Nat. Commun.7:13982 doi: 10.1038/ncomms13982 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Methods, Supplementary Figures, Supplementary ReferencesChemdraw file of chemical structures in Figure 4aChemdraw file of Figure 4a