Route optimization involves the reaction of PbI2 with excess HI to generate HPbI3, which subsequently undergoes reaction with excess CH3NH2 to deliver CH3NH3PbI3 thin films. High quality of intermediate HPbI3 and CH3NH2 abundance are two important factors to stable CH3NH3PbI3 perovskite. Excess volatile acid/base not only affords full conversion in nonstoichiometric acid–base reaction route but also permits its facile removal for stoichiometric purification, resulting in average efficiency of 16.1% in forward/reverse scans.Perovskite solar cells have received worldwide interests due to swiftly improved efficiency but the poor stability of the perovskite component hampers the device fabrication under normal condition. Herein, we develop a reliable nonstoichiometric acid–base reaction route to stable perovskite films by intermediate chemistry and technology. Perovskite thin-film prepared by nonstoichiometric acid–base reaction route is stable for two months with negligible PbI et al. develop a synthetic route using a nonstoichiometric acid-base reaction to prepare films stable in humid environments for two months.The stability of perovskite thin films and devices depends on a number of environmental factors, amongst which humidity. Here, Long  In terms of photovoltaic performance, it exceeds 22% power conversion efficiency in a short time, which is competitive with long-term developed thin-film-based solar cells 123456789111213111415Solution processed perovskite solar cells (PSC) using AMXPCE after 1,000 h under 10% one-sun illuminationet al.PCE with 1 cm2 large area. However, the stability of perovskite itself without shielding was still <1 week in a humid environment203/FAPbI3 perovskites were regarded to be unstable under high humidity. The degradation kinetics had been studied to some extent using thermal gravity analysis (TGA)3NH2 release was ambiguous and some believed that HI was released before CH3NH2 , probably due to poor electronic properties attributable to their long-chain cation and large-size anion components1718NH2 ref. . Besides °C refs , 25, but3NH3PbI3 perovskite, which revealed the role of the intermediate product as well as methylamine amount in achieving stability. Hybrid perovskite decomposes sequentially in terms of thermodynamics based on MA-recoverable degradation, such that it first loses CH3NH2 and then HI, leaving behind PbI2 solid. On the basis of this, we have developed an alternative two-step nonstoichiometric acid–base reaction route (NABR) for the synthesis of moisture-resistive perovskite, that is the production of starting HPbI3 using excess HI to react with PbI2, followed by perovskite conversion from HPbI3 using excess CH3NH2. We have established that CH3NH2 abundance in synthesis and the formation of high-quality HPbI3 built of columnar face-sharing PbI6 octahedra are two key parameters for stabilized perovskite. In NABR, excess and volatile reagents lead to complete reaction as well as stoichiometry, respectively. Therefore, a perovskite thin-film prepared via this route possesses reduced lattice vacancy, thus eliminating the penetration of undesirable H2O molecules into vacant sites and avoiding formation of monohydrate degradation product in association with H-bonding between H2O and MA+. We have demonstrated that the CH3NH3PbI3 perovskite thin-film remained highly stable in ∼65% humidity for up to 2 months with negligible PbI2-impurity, whereas other perovskites prepared by traditional one-step or two-step methods degrade distinctly after 2 weeks. The PSC using thin-film after humidity exposure delivered even better efficiency than that from freshly prepared film probably due to slight doping. This work provides an important insight into perovskite intrinsic stability and the utilization of simple chemical reaction for material control in PSC.In this work, we report our investigation of the degradation and recovery of CH3NH3PbI3, we have first investigated the degradation of traditional perovskites and used the recent defect-healing process for the recovery of degraded perovskite to check the transition products2 at 2θ=12.6° , (220) facets. The recovered perovskite films existed in the form of nanoscale crystals judging from the broad X-ray diffraction peaks. The photoluminescence mapping contrasts  , (100) and m crystal structure, and could be recovered to some extent (3NH3PbI3˙DMF (dimethylformamide), in which the MA+ is connected with solvent molecules (H2O/DMF) through H-bonding (To find a way to improve the stability of CH2θ=12.6° . Althoug2 vapour  right. Ton peaks  at 2θ=14ontrasts  for the 6°) refs , 25, 28.crystals , with phcrystals ; Fig. 1fe extent . The mon-bonding  and thus2 into 2–5 mg ml−1 MAI IPA solution for in situ observation of the recovery process via optical microscope. We found that after degradation, the yellow phase crystals did not differ much in morphology from that of the parent black perovskite  or (2) was right, which could yield CH3NH3PbI3˙DMF(3). The intermediate CH3NH3PbI3˙DMF  can be easily concluded from the final products. Degradation reaction (6) was apparently judged from CH3NH2-recovery in the glovebox (3NH2 molecules (6), and finally yielding a PbI2 solid and releasing HI/H2O vapour (7), although the kinetic pathways could produce different final products 233]− framework is the first important parameter, which can reduce the I-vacancy. Second, as CH3NH2 can recover the degraded perovskite through the reverse reactions of (5–7), CH3NH2 abundance is able to improve the stability of perovskite through retarding the degradation reaction. Therefore, the key to stable perovskite is to build a fully coordinated and robust [PbI3]− scaffold that accommodates CH3NH2 at A sites of perovskite AMX3 lattice, thus eliminating the inclusion of undesirable water molecules and suppressing the degradation reaction.Actually, reaction (5) has been verified through glovebox  and direΔrG1 and ΔrG2 are the changes of Gibbs free-energy for reaction (8) and (9), respectively.To synthesize stable perovskite, we propose a two-step route through NABR based on the above analysis where 3 that is stoichiometrically identical to the [PbI3]− framework of perovskite, permitting it to form perovskite without I-vacancy. In reaction (9), excess CH3NH2 allows complete conversion of HPbI3 to perovskite and thus eliminates the CH3NH2 vacancy. Different from the use of excess CH3NH3I to react with PbI2, both CH3NH2 and HI are facile for removal to yield vacancy-free perovskite due to their volatility at room temperature.In reaction (8), excess hydriodic acid promotes the reaction completely and yields HPbI3 crystal shows a hexagonal array of [PbI3]− anionic columns each composing of [PbI6] coordination octahedra that are stacked along their opposite triangular facets, with exact molecular formula HPbI3. Presumably, the protons in HPbI3 can move freely in the intervening space between hexagonal arrays of anionic coordination columns. Powdery HPbI3 as starting material for device fabrication was prepared following the literature procedure2O, DMF) and precipitate our products.  (3 had the advantage of solubility in much higher concentration (>2.5 M) than PbI2. We compared the absorption spectra of HPbI3, PbI2 and 1:1 CH3NH3I:PbI2 (one-step precursor) and found that those of HPbI3 and 1:1 CH3NH3I:PbI2 had 20–30 nm red-shifted absorption edge compared with that of PbI2, suggesting that coordination occurred between iodide and PbI2 in both HPbI3 and the 1:1 complex CH3NH3I:PbI2  . TherefoH3I:PbI2 . Moreoveid phase .The incrbI3 ref. . Due to ue-shift . This bl2 film prepared from DMF solution did not show the anisotropic rod-shape of its powders that should be essentially in accordance with the trigonal phase, but afforded uniform coverage and flatness on the substrates due to DMF coordination ) with perovskite3NH2 in NABR could reduce the crystal size , (101), (110) and (201) facets of hexagonal HPbI3, respectively. Even when the film was a little wet, it displayed the same indexed X-ray diffraction pattern as the dry films without/with baking after DMF loss, which is consistent with the single-crystal X-ray diffraction analysis on their solvate and dry crystal  peak at 12.6°  peak (3 directly precipitated by adding anti-solvents (such as diethyl ether) does not endure high humidity, probably due to HI/H2O residues.We have also checked the stability of starting HPbI01) peak . Except 01) peak . It has 3NH2 in this work. The best film remained stable for about 2 months in ∼65% humidity, without distinct PbI2-impurity from X-ray diffraction pattern and significant morphology change from freshly prepared samples  for nucleation. Therefore, it accelerated nucleation exponentially according to classical nucleation theory  and suppressed the Ostwald ripening in sequential nucleation, resulting in 100% film coverage by this key step  from X-ray diffraction monitoring. However, degradation beyond 2 months of the pin-hole-free film was quite different from that of the mesoporous as-prepared film without nucleation agent. We found a plausible trace of HPbI3 after 7 weeks at 2θ=11.6°, and then peaks at 2θ=8.1° and 8.7° indicating (001), (100) facets of CH3NH3PbI3˙H2O after 9 weeks, respectively. This can be explained by the sequential degradation thermodynamics in combination with degradation kinetics if we consider the surface effect. On the surface of pin-hole-free film, due to the ready release of both HI and CH3NH2, the sequential degradation reaction (5–7) occurred nearly simultaneously and thus only yielded PbI2 that we could observe. When degradation went to the interior that was single crystal-like3NH2 and HI were difficult to be released from the interior of high-quality of the film, thus as formed internal HPbI3 and CH3NH3PbI3˙H2O were detected by X-ray diffraction patterns after long-time degradation. This explanation has been further tested by the following experiments. First, we tried to convert the CH3NH3PbI3˙H2O back to CH3NH3PbI3 through heating  for 1–6 days without encapsulation of its high-efficiency photovoltaics, which indicates that its high stability primarily comes from device encapsulation. However, here we demonstrate that NABR using excess CH3NH2 to react with well-defined HPbI3 provides a reliable route to producing material-stable perovskites superior than those reported in previous work20Besides, it is notable that stable PSCs through layer shielding reported in previous works are generally lower than 16.2% efficiency in reverse scan203NH2 and HPbI3 are chemically stable and thus readily facilitate fabrication. Moreover, the synthesis of HPbI3 is much easier than CH3NH3I and can be collected using ethanol. Second, fundamentally speaking, the use of well-defined HPbI3 that is stoichiometrically identical to the intermediate complex as starting material is more chemically reasonable. Besides, we have fabricated a large area PSC up to 1.0 × 0.5 cm2 in area, which successfully delivered 15.0% PCE at the present stage. Thus, the present study demonstrates up-scaling potential for the assembly of modules in solar cells that function efficiently under ambient condition  coordination number to perovskite and CH3NH2 abundance as two important factors towards stable perovskite with as less site vacancies as possible. Excess and volatile acid/base leads to full coordination and stoichiometry, respectively, thus eliminating the penetration of water vapour and improving the stability in highly humid environments. The device has been optimized to 17.0/15.2% PCEs in forward/reverse scans after 1 month exposure in ∼65% humidity. This work provides an important insight into intrinsic stability and efficiency of perovskite as well as the utilization of simple reaction procedure with up-scaling potential via bottom-up synthetic chemistry for high-performance photovoltaics. Through the reaction demonstration, the vacancy-free insight into improving stability is probably a general paradigm for other perovskites. For example, FAxCsx1−PbIyBry3−, in which the FA+/Cs+ is also difficult to be removed, delivers as less vacancy as possible at the A sites of AMX3 perovskite for high stability. It is challenging to make MAPbI3 stable in traditional routes so far. Our NABR route provides a way to enhance the stability through smart reaction control to reduce vacancy without any other material composition. The intermediate structure of HPbI3 is especially interesting, in which the proton seems to move freely around the [PbI3] column. The perovskite after humidity test has even higher performance in this work, which may arise from protons in HPbI3.In conclusion, we have investigated the degradation and recovery of CH2 , N,N-dimethylformamide , spiro-OMeTAD (Merck), 4-tert-butylpyridine , Titania paste , Titanium(IV) isopropoxide , methylamine , hydroiodic acid , lithium bistrifluoromethanesulfonimidate , chlorobenzene , and all other chemicals were used as received without further purification.Methylammonium iodide , PbI3 preparation:HPbI HPbI3 powder was prepared by mixing PbI2 and excess HI (1.5:1 molar HI:PbI2) in DMF to ensure complete conversion, and stirring at 40 °C overnight. The light yellow precipitates were obtained by washing the precursor in abundant ethanol to remove excess HI until the supernatant turned to white. The excess HI and ethanol were then removed through filtration. The resulting powders were further dried and stored in an oven at 60 °C. It was then re-dissolved in DMF solution and different amounts of CH3NH2 ethanol solution were freshly added to obtain the NABR perovskite precursors. The resulting HPbI3 sample washed by diethyl ether did not exhibit high stability according to our control experiment. Needle-shaped semi-transparent HPbI3 solvate single crystals were grown by dissolving 1.5:1 molar HI:PbI2 in DMF followed by vapour diffusion of chlorobenzene into the mother liquor at 80 °C for 6 hours, yielding HPbI3 solvate crystal. After heating at 80 °C for 30 min, it turned faint yellow quickly and converted to HPbI3 crystal according to single crystal X-ray diffraction. The weight (HI+solvent):PbI2 ratio was 37.8% judged from TGA at 385 °C for single-crystal HPbI3 solvate, which was freshly collected from the mother liquor and placed on filter paper for 5 min, probably suggesting chemical formula HPbI3˙xDMF (x≤1) with disordered DMF inside the lattice  in weight loss after 60 °C. The HPbI3 powder recorded a 27.8% HI/PbI2 weight ratio at 350 °C by TGA  from a sealed-tube generator. Crystal data: hexagonal, a=b=8.7517 Å, 8.7339 Å, c=8.1802 Å, 8.1770 Å for HPbI3 solvate and HPbI3 with 1:3 atomic Pb/I ratio, respectively. The systematic absences are consistent with both noncentric space group P63mc (No. 186) and centric space group P63mcm (No. 193). As the proton has negligible X-ray scattering and the DMF molecule exhibits severe orientational disorder, structure determination was based on space group P63mc with only the Pb and I atoms subjected to anisotropic least-squares refinement using the SHELXL-97 program; 7,944 reflections measured, of which 509 are unique, Rint=0.099, R1=0.044, wR2=0.134 and GOF=1.05. The crystallographic data for this paper have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as No.1479488. These data can be obtained free of charge from CCDC via www.ccdc.cam.ac.uk/data_request/cif and supporting crystal information files (X-ray intensities of HPbIon files .2 (FTO) (TEC08) substrates were cleaned in an ultrasonic bath sequentially with acetone, 2-propanol and ethanol for 15 min, separately. The TiO2 precursor was prepared by 0.6 ml titanium isopropoxide and 0.15 ml 37%w/w HCl solution dissolved in 15 ml ethanol. The dense blocking layer TiO2 was coated onto FTO substrate by spin-coating of titanium precursor at 5,000 r.p.m. for 40 s, followed by annealing in air at 500 °C for 30 min. A 14 wt% solution of TiO2 nanoparticles in ethanol was spin-coated onto the dense TiO2 layer at 5,000 r.p.m. for 40 s to form a mesoporous scaffold and sintered in air at 550 °C for 30 min. After cooling to room temperature, the mesoporous TiO2 was immersed in aqueous 30 mM TiCl4 at 70 °C for 30 min, rinsed with DI water and annealed at 500 °C for 20 min. The perovskite precursor was prepared by mixing equimolar ratio of MAI and PbI2 in DMF by stirring at 60 °C. This solution was then spin-cast onto the TiO2 films at 3,000 r.p.m. for 30 s and annealed at 100 °C for 15 min to form the control CH3NH3PbI3 samples. For the perovskite precursors formed by HPbI3, 33 wt% MA solution in ethanol was mixed with 1.5 M HPbI3 DMF solution with increased volume of MA, and the fresh precursor was spin-coated on TiO2 at 2,500 r.p.m. and annealed at 100 °C for 30 min. With device optimization, we also used other concentration HPbI3 in association with excess MA and introduced nucleation agent  during the spin-coating at 10 s for full coverage. The spiro-OMeTAD was prepared by dissolving 75 mg spiro-OMeTAD with 25 μl LiTFSI solution (520 mg in 1 ml acetonitrile) and 35 μl 4-tert-butylpyridine in 1 ml chlorobenzene. Hole transport layer was deposited on the annealed perovskite film by spin-coating at 5,000 r.p.m. for 40 s to get the optimized efficiency. The devices were placed in a moisture-controlled cabinet overnight for oxidization of spiro-OMeTAD. Finally, 80 nm Au electrode was deposited by thermal evaporation with shadow mask area of 0.1 cm2. All device fabrication procedures were carried out in N2-purged glovebox.F-doped SnO2/perovskite/spiro-OMeTAD/Au and compared with freshly prepared films.For all the humidity stability studies, the perovskite films were spin-coated on FTO glasses, and the films were stored at room temperature (measured as 25±1 °C) in a controlled-humidity cabinet with a transparent glass door, with a low-power fluorescent light in the room. The relative humidity in the chambers was controlled to the desired humidity 65±5% by a beaker of water. And the air humidity in the room is floating from 60 to 75%. The relative humidity in the cabinet was precisely measured periodically throughout the stability experiments using a calibrated hygrometer. The cabinet was only opened just when taking sample out. As the humidity difference is very small, and the humidity cannot be greatly affected when opening the cabinet door and return to the desired humidity by the beaker of water through measurement. To emphasize the stability of perovskite itself and check the performance after humidity exposure, we deposited perovskite films without and with nucleation agent during spin-coating and then expose them in ∼65% humidity for 1–2 months. Humidity-exposed films were assembled into solar cells in the configuration of FTO/TiOα source. TEM imaging was performed on an FEI Tecnai Spirit microscope operating at 120 kV.The extinction and absorption spectra of solution samples were measured on a Hitachi U-3501 ultraviolet/visible/NIR spectrophotometer. The general images of the film morphology were obtained using an FEI Quanta 400 field emission scanning electron microscope  operated at 10 keV. X-ray diffraction measurements were performed with a Bruker D8 Advance Davinci powder X-ray diffractometer using a CuK2-filled glovebox under room temperature. Solar cell performance was scanned at scan speed 0.5 V s−1, dwell time 0.1 s, voltage step 0.05 V in forward and reverse scan loop. The scanning parameters related to the performance were attached  under simulated AM 1.5 sunlight generated by a 94011A-ES Sol series Solar Simulator. The solar cell devices were tested in Nattached . We usedThe authors declare that the data that support the findings of this study are available from the corresponding author on reasonable request.How to cite this article: Long, M. Z. et al. Nonstoichiometric acid–base reaction as reliable synthetic route to highly stable CH3NH3PbI3 perovskite film. Nat. Commun.7, 13503 doi: 10.1038/ncomms13503 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-25, Supplementary Tables 1-6.


An abrupt structural expansion is induced between 240 and 300 °C by hydrogenation. This structural modification deeply modifies the hydrogen sorption properties of the membrane, which indeed shows a strong reduction of the hydrogen capacity above 270 °C.Ni-Nb-Zr amorphous membranes, prepared by melt-spinning, show great potential for replacing crystalline Pd-based materials in the field of hydrogen purification to an ultrapure grade (>99.999%). In this study, we investigate the temperature evolution of the structure of an amorphous ribbon with the composition Ni The membrane splits the hydrogen molecule, and the atomic hydrogen is absorbed in the metal and diffuses towards the lower H2 concentration side. On the low-pressure face, the hydrogen atoms recombine to form H2 molecules and pure hydrogen is released thanks to the high selectivity of the metallic membranes. For the Pd-based membranes, H2 flux values in excess of 1 mol·m−2·s−1 have been reported [For purification of hydrogen to an ultrapure grade (>99.999%), crystalline Pd and Pd-Ag (100–200 μm thickness) membranes have been employed for several decades [reported .2 from CH4 or N2 [2 molecules do not need to split into hydrogen atoms to diffuse through the polymeric membranes [However, Pd is an expensive material and in certain conditions can suffer from embrittlement, due to the formation of hydrides. In order to prevent embrittlement, alloying of Pd with Ag, Au or Ru is usually performed . To mitiH4 or N2 . In thesembranes . Polymerembranes . Anotherembranes .−1, making them of great interest as potential Pd alternatives. Among other options, amorphous alloys formed from a combination of Ni and one or more early transition metals show great potential for this application [5 °C·s−1). One of the main concerns about such membranes is the possible crystallization during operation at high temperature, because it leads to the reduction in hydrogen diffusion pathways and often induces brittleness in the ribbons. Therefore, care should be used to avoid operation close to the crystallization temperature. In this framework, knowledge of the temperature evolution of the structure of the amorphous membranes is extremely important.Other classes of separation membranes are those based on ceramics or on porous carbon. The last one is brittle and has a selectivity for hydrogen in the range of 4–20 . Also, clication . Due thelication . Amorpho64Zr36 sample is on the order of 10−9 mol·m−1·s−1·Pa−0.5 [−9–10−8 mol·m−1·s−1·Pa−0.5 [A promising class of amorphous alloy membranes is the Ni-Zr or Ni-Nb-Zr alloys. Hara et al. reported that Ni-Zr-based alloys are stable and do not become brittle in the temperature range between 200 and 350 °C while the hydrogen permeability of the Ni1·Pa−0.5 ,15,16. M1·Pa−0.5 ,19,20,211·Pa−0.5 ,23, the 1·Pa−0.5 .2 can be formed [From a structural point of view, Ni-Nb-Zr membranes are quite complex. In their amorphous state, they are composed of icosahedral clusters with a random distribution of the three chemical elements on the vertexes ,24,25. De formed ,26,27. Fe formed .0.6Nb0.4)45Zr50Cu5 alloy, the copper substitution increases the permeability significantly. Yamaura et al. [−8 mol·m−1·s−1·Pa−0.5 for (Ni0.6Nb0.4)50Zr50 to 2.34 × 10−8 mol·m−1·s−1·Pa−0.5 for (Ni0.6Nb0.4)45Zr50Cu5, which is comparable to the permeability of conventional Pd-Ag alloys.Furthermore, Yamaura et al.  investiga et al.  showed t32Nb28Zr30Cu10 amorphous membrane, performed between 25 and 400 °C, by means of high temperature XRD measurements (HTXRD), either in a hydrogen or in a helium atmosphere. We will show that upon hydrogenation, the membranes display an abrupt expansion of the structure around 300 °C. In correspondence with this structural modification, the hydrogen absorption properties change and the membrane absorbs much less hydrogen in the high temperature regime than in the low temperature one.In order to obtain materials with high permeability but low hydrogen embrittlement, knowledge of their hydrogenation properties is important as it can provide information about the hydrogen effect on the material structure. In this framework, in this paper, we will report a study of the structure of a Ni32Nb28Zr30Cu10 sample. In the similar compound Ni42Nb28Zr30, which does not contain Cu, it has been reported that crystallization occurs at temperatures higher than 480 °C [As the occurrence of crystallization changes the absorption properties of the Ni-Nb-Zr membranes, we preliminary investigated the crystallization process by DTA measurements on the Nin 480 °C ,23,29; hThey present three subsequent thermally activated peaks, starting above 480 °C, centered around 520, 550 and 570 °C, when measured at ΔT/Δt = 15 °C/min. The inset of The hydrogen absorption properties of the membranes were measured by obtaining pressure-composition isotherms at different temperatures in the range between 158 and 400 °C; these temperatures are well below the crystallization temperature obtained by the previously reported DTA experiments.32Nb28Zr30Cu10 membrane are reported in 42Nb28Zr30). 32Nb28Zr30Cu10 membrane is higher at lower temperatures or, equivalently, the hydrogen solubility at a fixed p decreases as T increases; for example, for p = 1 bar one observes a hydrogen content of ~0.52 H/M at 158 °C and only ~0.27 H/M at 400 °C. Some single points along the pressure-composition isotherms were duplicated in order to verify the reproducibility of the measurements.The pressure-composition isotherms of the Ni32Nb28Zr30Cu10 membrane with the previous literature is possible. Indeed, in many cases Ni-Nb ribbons were hydrogenated by means of electrolytic charging [2 gas. Conic et al. [60Nb40, (Ni0.6Nb0.4)50Zr50 and (Ni0.6Nb0.4)70Zr30 [0.6Nb0.4)50Zr50 and ~0.7 mass % in (Ni0.6Nb0.4)70Zr30 [0.6Nb0.4)70Zr30 at 300 °C [0.6Nby0.4−Tay)x100−Zrx with y = 0, 0.1 and x = 20, 30, between 300 and 400 °C [0.6Nb0.4)70Zr30, the presently investigated membranes display a higher solubility by ~0.02 H/M at 300 °C and ~0.04 H/M at 400 °C.Only a limited comparison of the hydrogen absorption properties of the presently investigated Nicharging , instead4)70Zr30 . The hyd4)70Zr30 . Also Yat 300 °C . Recentld 400 °C . CompareThe sorption isotherms reported in hyd. However, the pressure-composition isotherms of the presently studied amorphous materials do not exhibit any plateau. In this case, one can still construct a van’t Hoff plot reporting ln(p) vs. 1/T, where p is the pressure at a fixed value of H/M at the various temperatures. Also, in this case, one can estimate the hydrogenation enthalpy, ΔHhyd, from the slope of the plot [32Nb28Zr30Cu10 membrane calculated for H/M from 0.30 to 0.42 in steps of 0.02 is reported in In the case of crystalline materials, for which a pressure plateau is observed in the p-c isotherms, the van’t Hoff plot reports ln(p) vs. 1/T, where p is the plateau pressure at temperature T (K) , and thethe plot . In ordehyd passes from 36 ± 1 kJ/mol at low T to 23 ± 1 kJ/mol at high temperature. It can be noted that ΔHhyd depends on the particular H/M considered, as reported in 0.6Nby0.4−Tay)x100−Zrx  membranes [0.6Nb0.4)70Zr30 the enthalpy decreases from 41 kJ/mol for H/M = 0.34 to 33 kJ/mol for H/M = 0.42 [The dependence of the hydrogenation enthalpy on the hydrogen content has already recently been reported for some other amorphous membranes for hydrogen purification, namely x100−Zrx membranes [hyd is much higher than that of the high temperature phase, showing the different sorption properties of the compound in the two temperature ranges. Indeed, in hyd obtained for the low T region. It is well evident that the experimental curve is shifted to a lower H/M by about 0.2–0.23.For the presently studied Niembranes . HoweverThe observed change of the absorption properties around 240 °C could be related to a structural modification of the sample. However, the membrane should retain its amorphous structure, at least within the detection limit of the previously reported DTA measurements, which do not show any peak in this temperature range. To further study this hypothesis, we performed an XRD study of the high temperature evolution of the sample structure.The XRD pattern of the pristine membrane, measured at room temperature in a helium atmosphere, is reported in 32Nb28Zr30Cu10 membrane was investigated by means of X-ray diffraction, heating the sample in a hydrogen (The temperature dependence of the structure of the Nihydrogen  atmosphe2 (32Nb28Zr30Cu10 membrane under a helium atmosphere (p ~ 1 bar) and the results are displayed in Heating the sample in H2 , the angThe angular position of the broad HTXRD peak is linked to the mean distance between atoms in the amorphous structure and the displacement towards lower 2θ values suggests an abrupt expansion of the structure at high temperatures.To obtain a more quantitative picture, it must be noticed that the peak position at the highest intensity represents the main average interatomic distance, in either clusters or atoms in the Ni-rich amorphous matrix.2 or He. In The 2θ values and the average interatomic distances obtained at the highest intensity are reported in 2 atmosphere. Moreover, in the present work, we show that the two phases, at low and at high temperature, present different hydrogen sorption properties, with the lower temperature phase displaying a much higher value for the hydrogenation enthalpy.From the present measurements, it is not possible to ascertain whether the entire icosahedra composing the amorphous structure or only some bonds between certain atoms expand in correspondence with the abrupt thermal expansion, but it is plausible that it may be due to an amorphous-to-amorphous phase transition. This transition would be induced by hydrogen, as it is not observed in the sample heated in He. An expansion of the Zr-Zr bond length induced by hydrogenation was already reported at room temperature , but in 32Nb28Zr30Cu10 was prepared by arc melting these metals in a purified argon atmosphere at AMES Laboratory Iowa, Ames, IA, USA. A melt spinning apparatus was used to fabricate the amorphous membranes at the CSIRO laboratory, Brisbane, Australia [Ni (purity 99.99% purity), Nb (99.8%), Zr (99.98%) and Cu (99.999%) were purchased from MilliporeSigma . An alloy button of Niustralia ,19. Typiustralia ,19.The X-ray diffraction patterns were obtained using a PANalytical X’Pert pro θ-θ diffractometer . The ribbons were not coated with Pd. This instrument was equipped with a heating stage with Anton Paar XRK 900 . Hydrogen gas was introduced into the heating chamber via a home-made volumetric apparatus. The sample was placed inside the chamber and first evacuated using the turbo pump by Pfeiffer Vacuum model number TSH071E .Simultaneous TGA-DTA measurements were conducted by means of a Setaram Sensys Evolution 1200 TGA system   under a The hydrogen absorption curves were recorded by the home-made Sieverts apparatus at Sapienza University of Rome described in Reference ,36. The 32Nb28Zr30Cu10 membrane are studied by means of pressure-composition isotherms and XRD measurements, respectively. In the temperature range between 242 and 300 °C, an abrupt decrease of hydrogen absorption is observed; correspondingly, the hydrogen concentration measured at 7 bar is reduced from ~0.6 H/M at 242 °C to ~0.45 H/M at 300 °C. The X-ray diffractograms confirm that the membrane remains amorphous also at the highest investigated temperature (400 °C), and clearly indicate an abrupt increase of the mean interatomic distance from ~2.31 Å at 200 °C to ~2.38 Å occurs. This abrupt expansion is evidenced only when the samples are heated in a hydrogen atmosphere, while a much lower and non-abrupt thermal expansion is observed when samples are heated in an inert atmosphere. Here, for the first time, an abrupt change of the physical properties of amorphous membranes when heated in hydrogen at high temperatures is reported. Incidentally, these findings suggest that we should avoid extrapolations from properties measured close to room temperature.The hydrogen adsorption properties and the amorphous structure of a Ni


Xa4 and Xa21 into MH725 and obtained an introgression rice line Wanhui 421 (WH421), which theoretically possesses 96.9% genetic background of MH725.Rice is a staple food crop in the world. With the increase in world population and economic development, farmers need to produce more rice in limited field. However, the rice production is frequently affected by biotic and abiotic stresses. The use of natural disease resistance and stress tolerance through genetic breeding is the most efficient and economical way to combat or acclimate to these stresses. In addition, rice with aromatic fragrance can significantly increase market value for its good grain quality. Mianhui 725 (MH725) is an elite restorer line that has been widely used to produce three-line hybrid rice in China. We previously introduced rice bacterial blight resistance genes Xa27 and Pi9, submergence tolerance gene Sub1A and aromatic fragrance gene badh2.1 in WH421 through backcrossing and marker-assisted selection. The newly developed introgression rice line was designated as Wanhui 6725 (WH6725), which theoretically possesses 95.0% genetic background of MH725. WH6725 and its hybrid rice conferred disease resistance to both blast and bacterial blight diseases and showed tolerance to submergence for over 14 days without significant loss of viability. Compared with non-aromatic rice MH725, WH6725 has strong aromatic fragrance. The major important agronomic traits and grain quality of WH6725 and its hybrid rice obtained in field trials were similar to those of MH725 and the control hybrid rice, indicating that WH6725 is as good as MH725 when it is used as a restorer line for three-line hybrid rice production.Here we report the introduction and pyramiding of disease resistance genes We have successfully developed a new restorer line WH6725 with disease resistance to rice blast and bacterial blight, tolerance to submergence and aromatic fragrance, which can be used to replace MH725 for hybrid rice production. Oryza sativa L.) is a staple food crop feeding more than three billion people. Rice yield had gone through two big leaps within a period of four decades from the 1960s to 1990s  genes against the infection by the two kinds of pathogens  genes is still an efficient and economic method for controlling diseases. Xa4, Xa21 and Xa27 are three dominant R genes in rice that provide race-specific and broad-spectrum resistance to X. oryzae pv. oryzae  whose function is to dampen ethylene production and gibberellic acid responsiveness during submergence, so as to economically use carbohydrate reserves and to prolong rice plants to submergence   resulted in the truncation of BADH2 enzyme in KDML105  is an indirect selection process in molecular breeding. A trait of interest is selected based on DNA-based molecular markers co-segregated with or derived from portions of the gene that controls the trait rather than the trait itself. MAS is a precise and efficient selection system that allows for recessive allele selection, early stage selection and multiple genes pyramiding without traditional phenotypic evaluation for each trait. The development of molecular markers for the selection of genes is a goal for many rice breeding programs , which theoretically possesses 96.9% genetic background of MH725  is an elite restorer line with good grain quality that has been widely used to produce three-line hybrid rice in China , 75-1-127(Pi9Pi9), IR64(Sub1ASub1A) or IRBB27(Xa27Xa27) followed by 4–5 rounds of backcrossing using WH421 as the recurrent female line  were selected for further backcrossing (data not shown). Theoretically, after 4–5 rounds of backcrossing, the BC4F1 and BC5F1 plants would have possessed 96.9 and 98.4% of the genetic background from the recurrent female parent WH421, respectively. In addition, the morphological phenotypes and growth duration of plants B4F1(badh2.1BadH2), B5F1(Pi9pi9), B5F1(Sub1Asub1A) and B5F1 (Xa27xa27) were similar to that of WH421 (data not shown). To combine the badh2.1 and Pi9 genes in a single plant, a B4F1(badh2.1Badh2) plant was crossed with a WH421 B5F1(Pi9pi9) plant to produce plant T29421  plant was crossed with a B5F1(Xa27xa27) plant to produce plant T14127  was then crossed with the plant T14127. Ninety-two F1 plants were produced from the cross. After genotyping, 6 of them were identified to carry heterozygous alleles at the badh2.1, Pi9, Sub1A and Xa27 loci . Theoretically, WH6725 possesses 95.0% genetic background of MH725. Therefore, we selected MH725 and its hybrid rice as the controls for WH6725 and its hybrid rice in the subsequent experiments.A two-step breeding approach was employed to introduce and pyramid the M. oryzae isolates collected from China  or the Philippines (11-17-1-2). MH725 was susceptible to ZB13, 11-3-1-1-2 and M39-1-3-8-1, whereas 75-1-127 and WH6725 were resistant to the 3 M. oryzae isolates  line II-32A and the thermosensitive-genic male sterility (TGMS) line 18892S, and their hybrid rice  were evaluated for rice blast resistance with X. oryzae pv. oryzae strains collected from 10 countries  and Thai R-7   , WH6725 and II-32A/WH6725 plants remained in light green  (viability = 83.3 ± 8.8%), WH6725 (viability = 82.2 ± 6.9%) and II-32A/WH6725 (viability = 84.4 ± 10.2%) plants recovered and survived after 14 days of submergence, whereas most of the MH725 (viability = 5.6 ± 5.1%), II-32A (viability = 8.9 ± 1.9%) and II-32A/MH725 (viability = 12.2 ± 3.8%) plants died, with a few plants survived but in poor health    (Table oC)  with medium grain shape  Table . Both WHC) Table . In addiC) Table . For hybC) Table . Both IIC) Table . They alC) Table . In addiC) Table . It shoubadh2.1 gene in backcrossing progeny as the badh2.1 gene controls the aromatic trait in recessive inheritance and the phenotypic selection cannot be done in heterozygous plants. Thirdly, MAS allowed us to pyramid the Xa4, Xa21 and Xa27 genes in a single rice line WH6725 without disease evaluation using R gene-specific X. oryzae pv. oryzae strains. Although the 3 dominant R genes are all for disease resistance to rice bacterial blight, each of them has its unique resistance specificity and their resistance spectrums are highly overlapped  is an introgression rice line that carries Sub1A in IR64 genetic background  marker NBS2-1, which was derived from the Pi9 gene (Table Sub1A gene was the co-dominant simple sequence repeat (SSR) marker RM23887 . The PCR reaction mixture of 20 μl consisted of 10 ng of rice genomic DNA, 0.15 mM of each dNTP, 0.15 mM of each primer, 2 μl of 10 × PCR buffer and 1 unit of Taq polymerase. Template DNA was initially denatured at 94 °C for 2 mins followed by 35 cycles of PCR amplification with the following parameters: 30 s of denaturation at 94 °C, 40 s of primer annealing at 52 °C for RM23887, 55 °C for 21 and RM224, 58 °C for M265 and M355, 60 °C for M124 and NBS2-1, and 1 min of primer extension at 72 °C. Finally, the reaction mixture was maintained at 72 °C for 5 mins before completion. M265 and M355 were amplified separately and mixed for each sample loading on agarose gel. The PCR products were electrophoretically resolved on a 1.5% agarose gel for NBS2-1, a 2.0% agarose gel for 21, M265 and M355, and a 3.5% agarose gel for markers RM23887, RM224 and M124 in 1 × TAE buffer.M. oryzae, ZB13, 11-3-1-1-2, 11-17-1-2 and M39-1-3-8-1, were used for inoculation experiments. The first three M. oryzae isolates were collected from Anhui, China. M39-1-3-8-1 was collected from the Philippines. M. oryzae isolates were grown on prune agar medium  at 28 °C in darkness for 7 days and at 26 °C under light for 3–5 days. The rice plants at 4-leaf-stage were inoculated with a spore suspension at the density of 1 × 105 spores/ml. The inoculated plants were immediately placed in a dew chamber for 24 h at 25 °C and 90% of humidity in darkness. The plants were then transferred to a growth chamber at 25–28 °C under a 12/12 h (light/dark) photoperiod and 90% humidity. Disease phenotypes were photographed at 7 days after inoculation.Isolates of X. oryzae pv. oryzae strains were grown on PSA medium  at 28 °C for 2 days. The bacterial cells were collected and suspended in sterile water at an optical density of 0.5 (OD600). Bacterial blight inoculation was carried out according to the leaf-clipping method  in the winter seasons (November–April) of 2013/2014 and 2014/2015, respectively. Field trial 3 and Field trial 4 were carried out in Hefei  in the summer seasons (May–October) of 2014 and 2015, respectively. In each field trial, paired parental lines and hybrid rice were arranged near to each other in the plots at the size of at least 12 mt-test for paired samples.The major important agronomic traits of rice, including growth duration, plant height, effective panicle number per plant, panicle length, total spikelet number per panicle, spikelet fertility, 1000-grain weight and yield per plant, were obtained from 10 plants grown in each plot and a total of 30 plants in 3 plots were measured for each variety. The growth duration was counted based on the number of days from sowing to 85% maturity per panicle in 90% of the plants population in a plot. The plant height was measured from soil surface to tip of the tallest panicle (awns excluded) at one day before harvest or at harvest. The number of effective panicle number per plant was the total number of panicles in a plant that produced more than 5 grains. The panicle length was measured from the base to the tip of a panicle (awns excluded). The total spikelet number per panicle contained the number of both filled and empty grains in a panicle. The spikelet fertility was the percentage of the number of the filled grains over the number of the total spikelet in a plant. The 1000-grain weight was measured with 1000 sun-dried filled grains. The yield per plant was the average weight of the filled grains per plants. P-value for each trait was calculated in Microsoft Office Excel 2007 according to a two-tailed t-test for paired samples.The grain quality was evaluated with the rice grains harvested from the four field trials mentioned above. Rice grain quality properties, including grain length, ratio of length/width, degree of chalkiness, amylose content, gel consistency, alkali spreading value and fragrance, were measured according to the methods described previously (Cruz and Khush


We demonstrate a polarizer-free electro-optical switch using dye-doped liquid crystal (LC) gels. The mechanism of dye-doped LC gels mainly involves the combination of polymer scattering and dye absorption. However, the domain size of polymer networks, dye concentration, LC concentration, and fabrication process can all affect the phase separation process and thus result in dye-doped LC gels with different electro-optical performance. We have studied experimentally the factors which can affect the dye-doped LC gels. The potential applications for dye-doped LC gels are flexible displays and electrically tunable light shutters. Most electro-optical switches using liquid crystals (LC) require at least one polarizer ,2,3. TheIn this paper, several factors affecting the electro-optical properties of dye-doped LC gels such as curing temperature, UV curing intensity, monomer concentration, and cell gap are discussed experimentally. Moreover, the optical analysis is also discussed. Some potential applications of dye-doped LC gels are flexible displays and electrically tunable light shutters.e = 1.6567, Δn = 0.1647 at λ = 589 nm; Δε = −5.7 at f = 1 kHz) and a M1 diacrylate monomer (bisphenol-A-dimethacrylate) and a dichroic dye S428  at 90:5:5 wt% ratios. The structure of the diacrylate monomer is as shown in The dye-doped LC mixture consists of a ZLI-4788 negative nematic liquid crystal  at a fixed temperature (10 °C) for 90 minutes. This fixed temperature for UV curing process is called the curing temperature. After photo-polymerization, chain-like polymer networks are formed along the z direction because LC directors are aligned along this direction by the vertical alignment layer, as shown in We then injected the mixture into an empty cell consisting of two glass substrates coated with a conductive layer of indium-tin-oxide (ITO) and a layer of polyimide (PI) without a rubbing treatment which is a mechanical process to create micro-grooves along the rubbing direction. The thicknesses of both layers are around 100 nm. The PI layer provides the vertical alignment for LC directors. The cell gap, the distance between two substrates, was 5 μm. The filled cell of the dye-doped LC mixture was then exposed to UV light  and decay time increases from 6 ms (10 °C) to 22.7 ms (40 °C). The reflectance at V=0 decreases from 55% (10 °C) to 42% (40 °C) with the increases of the curing temperature. That is because the better vertical alignment of LC directors, dye molecules and polymer networks at smaller domain size. The raising times are all around 0.4 ms at different curing temperature. The contrast ratio, a reflectance ratio of 0 V to 30 Vrms, is ~450:1 at 10 °C, 250:1 at 20 °C, 200:1 at 30 °C, and 300:1 at 40 °C. The contrast ratio decreases at T < 30 °C due to the larger polydomain and then increases as T > 30 °C caused by the dynamic scattering, a fluctuation of liquid crystal directors in polymer domains, which help decreasing the dark state reflectance. The reason why the larger domain has the dynamic scattering is still unclear.At V = 0, the cell shows a bright state because of the vertically aligned polymer networks, LC and dye molecules. At 30 VScanning Electron Microscopy (SEM) photographs after removing LC and dye molecules with hexane are shown in 2. The cell gaps were 5 μm. The reflectance as a function of a voltage is shown in th) increases from 4.82 Vrms (I ~ 0.354 mW/cm2) to 5.92 Vrms (I~2.6 mW/cm2 ). The reflectance at V = 0 increases from 44% (I ~ 0.354 mW/cm2 ) to ~57% ( I~2.6 mW/cm2). In 2) to 6.88 ms (I ~ 2.6 mW/cm2). The larger UV curing intensity results in smaller domain size of polymer networks. That causes the stronger anchoring energy and then enlarges the threshold voltage and boost the response time. The reflectance at V = 0 decreases under higher UV curing intensity owing to better vertically alignment at V = 0.To examine the effect of UV curing intensity (I), we prepared four samples with the same mixtures at the curing temperature 10 °C, but at different UV curing intensities, which were 2.6, 1.37, 0.733, 0.354 mW/cm2 at the curing temperature 20 °C. The cell gaps were still 5 μm. The voltage-dependent reflectance at different monomer concentrations is shown in In this section, the monomer concentration effect is discussed. We prepared three samples with different monomer concentrations of 3 wt%, 5 wt%, and 7 wt%. The fabrication processes of the samples were the same at the UV curing intensity 2.6 mW/cmrms (at 3 wt% M1) to 6.52 Vrms (at 7 wt% M1) with the monomer concentration due to the denser polymer networks. The reflectance at V = 0 decrease slightly (from 51% at 3 wt% M1 to 46% at 7 wt% M1). That is because denser polymer networks affect the vertical alignment of LC directors and also increase the scattering. CR increases from 222:1 (at 3 wt% M1) to 486:1 (at 7 wt% M1) owning to better scattering of higher monomer concentration at the high driving voltage. The threshold voltage increases from 2.1 V2. The cell gaps of two samples are 5 μm and 8 μm. As one can see in We can also increase the cell gap in order to enlarge the scattering and absorption of incident light. Two samples with a mixture of ZLI4788, M1, and S428 at 90:5:5 wt% ratios are made under the same curing condition:curing temperature ~30 °C and UV curing intensity ~2.6 mW/cmrms are shown in The images of a single pixel of the flexible polarizer-free electro-optical switch using dye-doped LC gels at V = 0 and V = 30 Vave(θ) is the average absorption coefficient, and βave(θ) is the average scattering coefficient. αave(θ) and βave(θ) satisfy the following equations:1 is the dye concentration, α// and α⊥ are the absorption coefficients when the polarization of incident light is parallel or perpendicular to the principal axis of dye molecule. ρ0 is the LC concentration, V is the average volume of a droplet. αeff(θ) can be expressed as:By considering the scattering and absorption, the reflectance (R(θ)) as a function of tilt angle (θ) of LC directors with respect to x-axis can be expressed as:// = 11.83 μm-1 and α⊥ = 0.962 μm-1 based on the experimental results. σeff in Equation (3) is the effective scattering cross section from all liquid crystal droplets and can be expressed as:We can estimate α0 is the geometrical optics cross section related to the domain size, δ is the polarization angle; Hve(θ) and Hvo(θ) stand for phase shift induced by e-ray and o-ray respectively. The averaged domain sizes of polymer networks are measured around 1.5 μm~3 μm.Based on anomalous diffraction approach ,13, scat1 = 0.05 g/cm3, and ρ0 = 0.89 g/cm3. α// = 11.83 μm-1 and α⊥ = 0.926 μm-1. The simulation result is shown in For numerical calculations we have chosen the following parameters according to the experiments: ρrms. The tradeoff is the bright state at 0 Vrms. The optical analysis agrees with the experimental results. The potential applications of dye-doped LC gels include flexible displays, electrically tunable iris, and electrically tunable band-pass filters, and electrically tunable light shutters.We have investigated the factors affecting the electro-optical properties of a polarizer-free electro-optical switch, the dye-doped liquid crystal gel. The mechanism of dye-doped LC gels mainly involves a combination of scattering and light absorption. The absorption results from the dichroic dye molecules. The scattering comes from the polymer network domains. The low curing temperature, strong UV curing intensity, and more monomer concentration result in good CR and fast response time because of large scattering caused by small domains. However, small domains cause large anchoring energy in liquid crystals. The driving voltage is then increased. Enlarging the cell gap to elongate the optical path of scattering and absorption is a way to improve the dark state at 30 V


N-trichloroacetylphosphorylamide (HL1) and dimorfolido-N-benzoylphosphorylamide (HL2) as representatives of carbacylamidophosphates were synthesized and identified by the methods of IR, 1H, and 31P NMR spectroscopy. In vitro HL1 and HL2 at 1 mM concentration caused cell specific and time-dependent decrease of leukemic cell viability. Compounds caused the similar gradual decrease of Jurkat cells viability at 72 h (by 35%). HL1 had earlier and more profound toxic effect as compared to HL2 regardless on leukemic cell line. Viability of Molt-16 and CCRF-CEM cells under the action of HL1 was decreased at 24 h  with no substantial further reducing up to 72 h. Toxic effect of HL2 was detected only at 72 h of incubation of Jurkat and Molt-16 cells .Dimorfolido-60 fullerene enhanced the toxic effect of HL2 on leukemic cells. Viability of Jurkat and CCRF-CEM cells at combined action of C60 fullerene and HL2 was decreased at 72 h  in comparison with the effect of HL2 taken separately.It was shown that C60 fullerene. More stable complexes are formed when DNA interacts with HL1 or C60 + HL2 structure. Strong stacking interactions can be formed between HL2 and C60 fullerene. Differences in the types of identified bonds and ways of binding can determine distinction in cytotoxic effects of studied compounds.In silico study showed that HL1 and HL2 can interact with DNA and form complexes with DNA both separately and in combination with C Nowadays, it is important to create new biocompatible nanomaterials that exhibit pharmacological properties, antitumor activity, and modulate the biological effects of chemotherapeutic drugs.Carbacylamidophosphates as the structural analog of β-diketones, that are promising class of biologically active compounds with antimitotic and antiproliferative activities, can be used as antitumor agents –3.N-(benzoyl)amidophosphate as representative of carbacylamidophosphates possesses an antitumor activity and interacts with DNA , DMSO .HL1 and HL2 were synthesized at the Department of Inorganic Chemistry, Chemical Faculty of Taras Shevchenko National University of Kyiv according to methods described earlier . The comUR-10 and a Perkin–Elmer Spectrum BX spectrometer on samples as KBr pellets. 1H and 31P NMR spectra in DMSO-d6 and methanol-d4 solutions were obtained on a Varian 400 NMR spectrometer at room temperature. Chemical shifts are referenced to SiMe4 as interior standard for 1H NMR and H3PO4 as exterior standard for 31P NMR.IR measurements were performed on a –1): ν = 3013 (NH), 1728 (CO), 1201 (PO); 1H NMR (DMSO-d6): 3.61 , 3.17 .HL1: m. p. 205 °C; IR : ν = 3100 (NH), 1685 (CO), 1200 (PO); 1H NMR (methanol-d4): 3.36 , 3.24 , 7.49, 7.56, 7.90  (8:8:5:1).HL2: m. p. 178 °C;  was synthesized in Technical University of Ilmenau (Germany) as described in [A highly stable aqueous colloid solution of Cribed in , 21.2 flasks at 37 °C with 5% CO2 in humidified incubator.The experiments were done on different human acute T cell leukemic cell lines. Cell lines were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures: Jurkat (ACC 282), CCRF-CM (ACC 240), and Molt-16 (ACC 29). The cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 2 mM gluthamine using 25-cm60 fullerene (10−5 M) during 1 h. After that, HL1 or HL2 was added to above medium and incubated for 24, 48, and 72 h.Cells in RPMI 1640 medium were preincubated with C60 fullerene was received as 100% (control sample contained 0.05 M DMSO).Cell survival without addition of chemical compounds and C5 cells) was placed into the 96-well microplates, 20 μl of MTT solution (4 mg/ml) was added to each well, and the plates were incubated for another 2 h. The culture medium was then replaced with 100 μl of DMSO; diformazan formation was determined by measuring absorption at 570 nm with a microplate reader Tecan Infinite M200 Pro (Switzerland).Cell viability was assessed by the MTT reduction assay . At indi60 fullerene have been studied. We took into consideration the following structures of DNA molecule: 2MIW , 1XRW , and 2M2C . We applied the algorithm of systematical docking (SDOCK+), built-in the QXP package ) [60 fullerene, we generated 300 potentially possible complexes with DNA, the 10 best of which were selected for the next stage, using a scoring function, built-in the QXP package [The double-helix DNA molecule was used as a template from PDB (Protein Data Bank) base. The interactions of DNA molecule with HL1 or HL2 separately and in combination with C (Rmsd)) . To each package .60 fullerene were characterized by the following parameters: (1) the number of hydrogen bonds, (2) the area of contacting surfaces of DNA and corresponding structure, (3) the distance between the DNA and docked structure, and (4) the total energy of the binding structure.The interactions of the DNA molecule with HL1 or HL2 separately and in combination with the C60 fullerene, we conducted the short-molecular dynamics  using a Nosé-Poincaré-Anderson algorithm (NPA) [To assess the stability of the complexes of chemical compounds with Cmics MD,  ps usinghm (NPA) . The calhm (NPA) .p < 0.05 was considered statistically significant. Data processing and plotting were performed by IBM PC using specialized applications GraphPad Prism 7 .The data were represented as M ± SD of more than five independent experiments. Mean (M) and standard deviation (SD) were calculated for each group. Statistical analysis was performed using two-way ANOVA followed by post Bonferroni’s tests. A value of anti-positions to each other as in most carbacylamidophosphates. In the structure of HL1, the bond lengths C–O (1.202(4) A) and C–N (1.346(4) A) are influenced by the substituent nature near the carbonyl group and have typical values for trichloracetylamide derivatives. The carbon atom of CCl3 group has a tetrahedral environment  group reflects in the shortening of intramolecular hydrogen bonds N···O and O…H type: for HL1—1.81(1) A and 2.73(1) A, and for HL2—2.103(3) A and 2.946(3) A, respectively.More electronegative character of CClHL1 and HL2 at 1 mM concentration were screened for their toxicity against human T cell leukemic cells Jurkat, CCRF-CEM, and Molt-16 using MTT assay.It was shown that HL1 and HL2 decrease the viability of leukemic cells. The observed toxic effect was cell specific and time dependent  as compared with Molt-16 cells (37% at 72 h)  only in 72 h, while no substantial decrease of CCRF-CEM cell viability was detected , HSG (submandibular gland), and HL-60 (promyelocytic leukemia) as well .It was shown that β-diketones exist mainly in the enolic form and form metal chelates with Fe, Cu, and Zn ions. Recent studies suggest that metal chelates induce apoptotic cell death in various tumor cell lines and they are potential antitumor agents against malignant melanomas , 31.60 fullerene on leukemic cells was investigated.To estimate the possibility to enhance the toxicity of studied compounds, their combined action with C60 fullerene (10-5 M) does not affect the viability of leukemic cells during incubation period (data not shown).It was shown that C60 fullerene (data not shown).No increase of HL1 toxicity was detected when Jurkat, Molt-16, or CCRF-CEM cells were preincubated with C60 fullerene and HL2, an enhanced toxic effect in comparison with the effect of HL2 taken separately was observed. In this case, a viability of Jurkat and CCRF-CEM cells was additionally decreased by 20 and 24% at 72 h, respectively .60 fullerene was studied depending on the sequence of interactions. If initially C60 fullerene binds with DNA, and then HL1 or HL2, the formed structure is designated as C60 + HL1 or C60 + HL2; otherwise—HL1 + C60 or HL2 + C60. We considered two options of the binding-studied structures with DNA molecule, namely with small and large grooves, and also the possible intercalation of these structures into the small groove  and C60 + HL1 (6.42 Å) are also observed. Due to such shift of C60 molecule, a partial displacement of HL1 compound  from the intercalation site  , and in a large groove—8.56 Å (for HL2—3.85 Å). However, a complete break of stacking interaction between C60 fullerene and HL2 does not happen.HL2 + Cves Fig. a, b. So,60 fullerene forms the strong stacking interactions with HL2 and nitrogenous bases of DNA double helix , and in the case of C60 + HL2—2.7 Å (for HL2—3.29 Å). In addition, at such intercalation, there is no displacement of chemical compound from the original nucleotide environment.At intercalation of the studied structures into a small groove of DNA, the Clix Fig. c, d. So,60 + HL1 or C60 + HL2 intercalation into DNA molecule are lower for C60 + HL1 structure than for HL1 + C60 or HL2 + C60 structures. The Bump value for C60 + HL1 structure is 17.1 kJ/mol and Int—4.2 kJ/mol, while for HL1 + C60 structure, these parameters are higher—19.6 and 7.2 kJ/mol, respectively. These data allow to suggest that the studied chemical compounds form more stable structures with C60 fullerene when C60 molecule is initially bound to DNA.As shown in Table 60 fullerene, one can suggest the formation of cation-π bonds between HL1 and C60 fullerene due to CCl3 group  and dimorfolido-N-benzoylphosphorylamide (HL2) at 1 mM concentration in the cellular medium caused the decrease of cell viability, which value was dependent on the cell type and duration of incubation. HL1 and HL2 caused the similar gradual decrease of Jurkat cells viability. HL1 had earlier and more profound toxic effect as compared to HL2 both on Molt-16 and CCRF-CEM cells. Toxic effect of HL2 was detected only at 72 h of Jurkat and Molt-16 cells incubation.We have estimated effect of two carbacylamidophosphate derivatives with different substituents on the viability of leukemic cells of three lines. Dimorfolido-60 fullerene facilitated the toxic effect of HL2 on Jurkat and CCRF-CEM cells at 48 and 72 h.It was shown that C60 fullerene were studied. The chemical compounds formed the stable complexes with DNA: HL1 when binding with a great groove and at intercalation into a small groove, while HL2 only when bound with a small groove. At the combined action of HL1 or HL2 with C60 fullerene, the C60 + HL2 structure formed the stable complex with DNA when bound with a small groove and at intercalation into it, while the C60 + HL1 structure formed the stable complex with DNA only in one case—when binding with a large groove. In this case, the strong stacking or cation-π interactions can be formed between these chemicals and C60 fullerene. Thus, the chemical compounds form the stable complexes with DNA both individually and in combination with C60 fullerene, but the differences in the types of bonds and ways of binding could be the cause of their different cytotoxic effects.By use of computer simulation, the interactions of DNA molecule with HL1 or HL2 separately and in combination with C


The prolyl-3,4-dihydroxylase Ofd1 and nuclear import adaptor Nro1 regulate the hypoxic response in fission yeast by controlling activity of the sterol regulatory element-binding protein transcription factor Sre1. Here, we identify an extra-ribosomal function for uS12/Rps23 central to this regulatory system. Nro1 binds Rps23, and Ofd1 dihydroxylates Rps23 P62 in complex with Nro1. Concurrently, Nro1 imports Rps23 into the nucleus for assembly into 40S ribosomes. Low oxygen inhibits Ofd1 hydroxylase activity and stabilizes the Ofd1-Rps23-Nro1 complex, thereby sequestering Ofd1 from binding Sre1, which is then free to activate hypoxic gene expression. In vitro studies demonstrate that Ofd1 directly binds Rps23, Nro1, and Sre1 through a consensus binding sequence. Interestingly, Rps23 expression modulates Sre1 activity by changing the Rps23 substrate pool available to Ofd1. To date, oxygen is the only known signal to Sre1, but additional nutrient signals may tune the hypoxic response through control of unassembled Rps23 or Ofd1 activity. Animals, plants, and fungi need oxygen to release energy within their cells and for other chemical reactions. Enzymes that use oxygen typically become less active when less oxygen is available, and this makes them well suited to help cells sense oxygen. These enzymes include oxygenases, some of which modify proteins by adding oxygen to specific sites in a reaction called hydroxylation. Oxygenases control how mammals adapt to low levels of oxygen – a condition referred to as hypoxia. These enzymes achieve this by hydroxylating a protein – specifically a transcription factor – that turns on genes for survival in low oxygen. Cells quickly destroy the hydroxylated transcription factor but when oxygen is limiting, it remains unmodified. This means that, rather than being destroyed, the transcription factor binds DNA, and activates genes that keep the cells alive and growing in low oxygen.In fission yeast, an oxygenase called Ofd1 controls the activity of a transcription factor called Sre1. Yeast requires Sre1 to grow when oxygen is limiting. Exactly how Ofd1 regulates Sre1 is unknown, but the mechanism is different from that in mammals because regulation of gene expression does not need Sre1 to be hydroxylated.Now, Clasen et al. report that Ofd1 actually hydroxylates another protein called Rps23. This protein is one of about 80 that form the cell’s protein-building machinery, the ribosome. It turns out that, before Rps23 becomes part of the ribosome, it binds Ofd1 in a complex with other proteins. The multi-protein complex then acts to hydroxylate and transport Rps23 into the nucleus, where ribosomes are built and where the cell stores its DNA. When little oxygen is around, Ofd1 cannot hydroxylate Rps23. This stops the complex from falling apart and traps Ofd1 away from the transcription factor Sre1. When not bound by Ofd1, Sre1 is free to turn on genes that allow growth at low levels of oxygen. Finally, Clasen et al. show that more unassembled Rps23 means less Ofd1 is available to inhibit Sre1, which controls the yeast cell’s response to hypoxia.Humans have proteins similar to Ofd1 and Rps23. As such, this pathway for sensing oxygen in yeast may occur in humans too. Further work is now needed to explore if other enzymes that hydroxylate ribosomal proteins work in a similar way. Eukaryotic cells require oxygen and must adapt to changes in its supply to maintain homeostasis. Research on mechanisms underlying oxygen homeostasis has focused mainly on hypoxia-inducible factor (HIF) signaling in mammals . HIFs arSchizosaccharomyces pombe. Sre1 is a sterol regulatory element-binding protein (SREBP) homolog required for adaptation to low oxygen environments  in response to sterol depletion during hypoxia  and fly (Sudestrada1)  . uS12 isstrada1) . While tstrada1) . This smS. pombe, as a binding partner and substrate of Ofd1 and investigated the role that P62 dihydroxylation plays in fission yeast. We confirmed that Ofd1 dihydroxylates Rps23 and further report that Ofd1 and Rps23 form a complex with Nro1. This complex functions to transport newly synthesized Rps23 to the nucleus while simultaneously facilitating the dihydroxylation of P62. In addition, Ofd1 activity regulates formation of the Ofd1-Rps23-Nro1 complex such that decreased enzyme activity under low oxygen stabilizes the complex. As a result, unassembled Rps23 regulates Sre1N signaling by sequestering Ofd1 in an oxygen-dependent manner, thereby coupling hypoxic gene expression to rates of ribosomal synthesis. Finally, we identified a conserved Ofd1 binding sequence shared by all known Ofd1 binding partners. This study outlines a new paradigm for control of hypoxic adaptation, assigns a second function to uS12 hydroxylation, and defines a distinct mechanism by which an oxygenase functions as a cellular oxygen sensor.Here, we independently identified uS12, known as Rps23 in RPS23 gene) as a binding partner of OGFOD1 and confirmed that the interaction is conserved between Ofd1 and Rps23 in fission yeast  lysine, mixed, and processed to enrich for Rps23 from assembled 40S subunits. Following digestion by LysC, H/L peptide ratios were measured by LC-MS/MS  mass shift for mammalian RPS23 aa 61–68: QPNSAIRK . P62 is LC-MS/MS . We deteLC-MS/MS . Analysified P62 . We foun2A D144A . CollectWhile Rps23 is the only known enzyme substrate of Ofd1, we previously showed that Ofd1 directly binds Nro1, a negative regulator of Ofd1 and a nuclear import adaptor . To dete+rps23 and +rps2302 – code for identical Rps23 proteins. While deletion of either gene results in a modest decrease in total Rps23 levels, levels of unassembled Rps23 are dramatically reduced by ~90% -labeled LC-MS/MS to quantify P62 hydroxylation. The ratio of Ofd1:Rps23:Nro1 (1:10:10) replicated the physiological ratio of Ofd1:unassembled Rps23:Nro1 in cells (data not shown). Consistent with the in vivo results, addition of Nro1 to the in vitro reaction increased Rps23 P62 dihydroxylation by 1.6 fold . These data demonstrate that Rps23 is a specific positive regulator of Sre1 signaling.To test if Rps23 regulates Sre1, we first analyzed growth of mediates . Cobalt chloride . Growth chloride . Consistof Sre1N . The fain cobalt . Furthers normal . Finallysitivity , and we sitivity . These tsre1 locus, bypassing the requirement for cleavage , a cell permeable competitive inhibitor of Ofd1 that is metabolized to a 2OG analog, to test if enzyme activity affects Ofd1 binding to Rps23 and Nro1 . Ofd1 imIn this study, we report three major findings: (1) Nro1 functions in a new nuclear import pathway for the small ribosomal protein uS12/Rps23; (2) Nro1 is the first protein adaptor for prolyl dihydroxylation; and (3) Rps23 has an extra-ribosomal function essential for the fission yeast hypoxic response.Unassembled Rps23 sits at the center of these findings, and our data support the following model for how Rps23 is incorporated into ribosomes: Nro1 binds newly synthesized Rps23 in the cytosol; Ofd1 then binds Rps23-Nro1 and dihydroxylates P62 during nuclear import; Nro1 releases dihydroxylated Rps23 in the nucleus where it is incorporated into assembling 40S ribosomal subunits. Multiple lines of evidence support this model. Rps23 binds Nro1 in vitro, the interaction does not require Ofd1, and binding is stable in the absence of chemical crosslinker , suggestOfd1 binding to Rps23 and Nro1 is mediated through a shared binding sequence in vitro . Given pnro1Δ cells is unknown. Previous studies showed that prolyl-3-hydroxylation is sufficient to restore the defects in translation termination caused by loss of the Ofd1 homolog Tpa1, suggesting that loss of this post-translational modification underlies the translation defects  was purchased from Cambridge Isotope Laboratories . DMOG was from Frontier Scientific Inc . Protease inhibitors  were included in buffers where indicated.Common lab reagents were obtained from either Sigma or Thermo Fisher Scientific. Oligonucleotides were provided by Integrated DNA Technologies . Heavy lysine , 3% [w/v] glucose, 225 μg/ml each of uracil, adenine, leucine, histidine, and lysine) to 1 × 107 cells/ml unless otherwise indicated  under control of the constitutive CaMV promoter was described in +rps2302 and rps2302 point mutants were cloned into a constitutive adh1-driven expression vector with +leu2 marker, derived from pART1 , mouse anti-GFP monoclonal , mouse anti-Rps23 monoclonal , mouse anti-Rps5 monoclonal , and mouse anti-actin monoclonal . Lab-made antibodies are rabbit polyclonal antisera and were previously described: anti-Ofd1 : IRDye800CW/IRDye680RD mouse and rabbit IgG, IRDye 680RD Detection Reagent. Commercially obtained antibodies include mouse anti-GST monoclonal (RRID:RPS23 represented 80% of positive clones (56/70). Confirmation of screen hits was performed following Matchmaker Gal4 Two-Hybrid System user manual  using the yeast two-hybrid S. cerevisiae strain AH109 (Yeast two-hybrid screen (>3 fold library coverage) was conducted at Duke University using a human fetal brain cDNA library. in AH109 . Briefly7 cells/ml. Cells (1 × 108) were pelleted and lysed by vortexing with glass beads (0.5 mm) for 10 min at 4°C in 1% [v/v] NP-40, 50 mM Hepes-HCl pH 7.4, 100 mM NaCl, 1.5 mM MgCl2, and protease inhibitors. Lysates were centrifuged at 20,000 × g for 1 min at 4°C and the resulting supernatant was designated the whole cell lysate (WCL). Ribosomes were pelleted from WCL by centrifugation at 80,000 rpm for 16 min at 4°C using an Optima TLX ultracentrifuge and TLA100 rotor . WCL (5 μg) and ten-fold by volume supernatant were loaded on 16% acrylamide gels. Rps23 detected in the ribosome-cleared supernatant by immunoblot was considered unassembled and normalized to Dsc5. Rps23 fractions were calculated as the mean of three biological replicates ± SEM and analyzed by paired, one-tailed t test.Strains were cultured in rich medium to 0.5–1.0 × 10E. coli BL21-CodonPlus (DE3)-RIPL competent cells  with 0.1 mM IPTG at 37°C for 2 hr in LB medium. Cells were sonicated in lysis buffer  prior to the addition of 1/10 vol 10% [v/v] Triton X-100. Lysates were incubated for 10 min at 4°C then centrifuged at 20,000 × g for 10 min. MagneGST particles  were blocked with 1% [w/v] BSA diluted in lysis buffer for 30 min at room temperature then incubated for 1 hr at room temperature with saturating amounts of cleared cell lysates. Following 3 washes in 25 mM phosphate pH 6.8, 250 mM NaCl,1% [v/v] TWEEN 20, and protease inhibitors, the particles were resuspended in 200 μl containing 84.5 nM Ofd1 or 84.5 nM Nro1 diluted in lysis buffer plus 1% [v/v] Triton X-100 for 1 hr at 4°C. Ofd1 and Nro1 were purified under native conditions as previously described  using BamHI/NotI restriction sites. Fusion protein expression was induced in escribed . ParticlStructures were downloaded from the PDB  and anal8 cells (Nro1 IP) or 2 × 108 cells (Ofd1 IP) were pelleted, washed in PBS  and resuspended in 1 ml PBS plus 2 mM DSP  or vehicle (8% DMSO unless stated otherwise) for 5 min to crosslink proteins. The reaction was quenched by addition of 1 M Tris-HCl pH 7.5 to a final concentration of 20 mM Tris. Ribosome-cleared lysates were generated as described above and 1.0–1.5 mg protein in 600 μl volume was incubated with affinity-purified antibodies (8 ng antibody/μg protein) and 30 μl Protein A agarose beads  for 2 hr at 4°C. Beads were washed three times and resuspended in SDS lysis buffer  plus protease inhibitors prior to boiling  and immunoblotting.Ofd1 and Nro1 immunopurifications (IPs) were performed as previously described  with mod2) plus protease inhibitors and 1 mM DTT, and lysed by vortexing with glass beads (0.5 mm). Lysates were cleared by centrifugation at 20,000 × g for 10 min and heavy-labeled or 1:1 mixtures of heavy- and light-labeled proteins were layered over a 10% sucrose cushion  plus protease inhibitors. Lysates were centrifuged at 41,000 rpm for 6 hr in the SW 41 Ti Rotor (Beckman Coulter) and ribosome pellets were solubilized in 150 μl of 6 M ultrapure urea, 3 M LiCl, 50 mM KCl, and 5 μM BME (pH adjusted to 4.5) overnight at 4°C using micro stir bars. Lysates were cleared by centrifugation at 72,000 rpm for 1 hr in the TLA100 rotor, and proteins were precipitated from the supernatant overnight with the addition of two volumes of 20% [w/v] TCA. Precipitated proteins were washed with 1:1 [v/v] ether:ethanol and air-dried prior to resuspension in 8 M ultrapure urea, 150 mM Tris-HCl pH 8.5. Samples were separated by gel electrophoresis using 16.5% acrylamide gels  and stained with Super Blue . 15–20 kDa gel bands were excised and analyzed by LC-MS/MS.Strains were cultured for ≥15 generations in SILAC medium  as described . PelleteGel pieces were destained and rehydrated in 150 μl 0.01 µg/µl LysC  in 25 mM triethylammonium bicarbonate (TEAB) then covered with an additional 150 μl of 25 mM TEAB and digested overnight at 37°C. Peptides were extracted from the gel pieces with 50% [v/v] acetonitrile, 0.1% [v/v] trifluoroacetic acid (TFA) and evaporated to dryness in a speed vac. Samples were rehydrated in 0.1% [v/v] TFA and loaded on Oasis HLB µElution solid phase extraction plates  and desalted with two 100 μl aliquots of 0.1% [v/v] TFA followed by 100 μl of 10 mM TEAB. Each sample was then step fractionated under basic conditions with 10%, 25%, and 75% [v/v] acetonitrile in 10 mM TEAB yielding three fractions for each of the samples for LC/MS/MS analysis. These fractions were then dried and brought up in 10 μl of 2% [v/v] acetonitrile, 0.1% [v/v] formic acid.Schizosaccharomyces pombe using the Mascot  search engine running through Proteome Discoverer v.1.4 (Thermo Scientific). The precursor mass tolerance was set to 15 ppm and the fragment tolerance was set to 0.03 Da. For the search settings, the following modifications were set as variable: deamidated , oxidation , dioxidation (P), and K8 Heavy (K). The K8 Heavy modification adds a mass of 8.014 Da, and SILAC pairs within four ppm mass were considered for calculating SILAC ratios.The LC-MS/MS analysis was performed on a Q-Exactive HF mass spectrometer  with a nanoACQUITY nano flow chromatography system (Waters). The samples were trapped at 5 μl/min then eluted onto a 20 cm x 75 µm i.d. C18 column for a 90 min gradient at 300 nl/min. The mass spectrometer settings were 240,000 resolution for MS and 35,000 resolution for MS2 with target of 3e6 for MS and 1e5 for MS2. Maximum injection times were set to 60 and 300 millisec respectively and a normalized collision energy of 28 was used. The data from the three fractions for each sample were combined and searched against the RefSeq2015 All SILAC data were filtered for unique, high confidence peptide spectrum matches (PSMs). For quantification, missing quan values were replaced with the minimum intensity and single-peak quan channels were used. To quantify Rps23 P62 hydroxylation between SILAC pairs, first the labeling efficiency  multiplied by 100, with %L equal to 100 – (%H).nro1Δ cells, we assumed that all Rps23 P62 is unmodified in ofd1Δ cells. This assumption was based on the finding that over 99.9% of the dihydroxylated P62 in the wild-type-ofd1Δ SILAC pair originated from the heavy-labeled wild-type cells , where H/L is the median ratio of unmodified P62 in the ofd1Δ (H) - nro1Δ (L) SILAC pair. From this analysis, we found that unmodified P62 accounts for 32% of Rps23 in nro1Δ ribosomes. We used the same logic to calculate the percentage of dihydroxylated P62 in nro1Δ cells using the wild-type-nro1Δ SILAC pair and assuming wild-type cells contain only dihydroxylated P62. Given conservation of mass and the expectation that P62 exists in one of only three states, we then estimated the relative percentage of monohydroxylated Rps23 P62 in nro1Δ cells as 100 - (% unmod) – (% di), leading to the conclusion that approximately 60% of Rps23 P62 is monohydroxylated.To determine the relative abundance of the unmodified and monohydroxylated forms in pe cells . In addi+S. pombe rps23 cDNA was cloned into pMAL-c5X expression vector , and the plasmid was transformed into BL21-CodonPlus (DE3)-RIPL cells. The transformants were grown to OD600 = 0.6 at 37°C and then induced with 0.4 mM IPTG overnight at 18°C. Cells were harvested by centrifugation and cell pellets were stored at −80°C. Bacterial pellets were resuspended in lysis buffer  and lysed by French press. Cleared lysates were loaded onto an amylose resin column (NEB) and MBP-Rps23 was eluted with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM maltose, 1 mM EDTA, and 0.5% [v/v] Triton X-100. Fractions containing MBP-Rps23 were pooled and dialyzed in 20 mM Tris-HCl pH 8, 1 mM EDTA, and loaded onto a Mono Q anion exchanger . Sample was eluted with 20 mM Tris-HCl pH 8, 1 M NaCl, and purified MBP-Rps23 was collected in the flow-through, concentrated, and stored in 20 mM Tris-HCl pH 8, 150 mM NaCl.4, and 0.3 mM 2OG. After incubation at 37°C for 1 hr, the reaction was terminated by the addition of 1.25 μl 2 M HCl and analyzed by LC-MS/MS.For TMT analysis, reactions were performed in 20 μl volumes with Ofd1 (0.5 μM) and MBP-Rps23 (5 μM) diluted into lysis buffer  with and without Nro1 (5 μM). Ofd1 and Nro1 were natively purified as previously described . Reactio4, and 0.3 mM 2OG. The reaction was incubated overnight at 37°C.For the initial identification of hydroxylated P62, the reaction volume was scaled up to 50 μl. Ofd1 (5.2 μM) and purified human 6xHis-RPS23 (79 μM) were diluted into the lysis buffer along with 0.1% [w/v] BSA, 0.5 mM DTT, 4 mM ascorbate, 0.15 mM FeSOReaction samples were LysC-digested and labeled with TMT reagents (Thermo Fisher Scientific) for 1 hr at room temperature. Samples were step fractionated with four basic, reversed phase fractions of 5%, 15%, 25%, and 75% [v/v] acetonitrile in 10 mM TEAB, and run on a Q Exactive HF mass spectrometer (Thermo Scientific). The mass spectrometer settings were 120,000 resolution for MS and 60,000 resolution for MS2 over a 90 min gradient, targeting 3e6 ions for MS and 1e5 for MS2. Maximum injection times were 100 millisec for MS and 200 millisec for MS2. A stepped normalized collision energy 30/35 was used for fragmentation. The isolation window was set to 1.2 Da with a 0.5 offset, and the instrument was set to fragment the top 15 peptides by data dependent analysis with a dynamic exclusion of 10 s. An inclusion list  was inserted to give preference to the QPNSAIRK peptide over the highest abundance peptides. The masses in the inclusion list represented the various modification states of P62 , as well as the TMT label and deamidated N (+0.98 Da). To increase the signal for the dihydroxylated P62 peptide, the 75% fraction was re-run with the settings ‘Do Not Pick Others.’Schizosaccharomyces pombe using the Mascot (Matrix Science) search engine running through Proteome Discoverer v.1.4 (Thermo Scientific) with one missed cleavage allowed and a tolerance of 10 ppm MS and 0.03 Da MS2.Data was searched against the RefSeq2015 database In order to compare the relative abundance of Rps23 P62 hydroxylation in the absence and presence of Nro1, first the relative abundance of Rps23 protein between the two samples was calculated using non-P62-containing peptides. PSMs with unique QuanResultIDs were used for quantification. The intensities from each PSM  and vehicle (0.5% [v/v] ethanol) in rich medium for 1 hr. For live-cell imaging, 5 × 106 cells were pelleted, resuspended in 15 μl rich medium, and 1 μl spotted on untreated glass slides immediately prior to imaging. Cells were imaged on a Zeiss Axio Imager M2 upright fluorescence microscope . Images were captured using a Hamamatsu ORCA-ER digital camera (Japan) and iVision software , and brightness/contrast settings were adjusted using ImageJ such that the range was identical across the strains under comparison.Indicated strains were cultured in rich medium to 4–7 × 102 400 workstation . Briefly, cells grown to 1 × 107 cells/ml were resuspended in oxygenated or deoxygenated rich medium at 5 × 106 cells/ml for 3 hr then harvested and flash froze in liquid nitrogen. Cell pellets were lysed in 27 mM NaOH, 1% [v/v] 2-mercaptoethanol followed by TCA precipitation. Protein pellets were resuspended in SDS lysis buffer, and 20–100 μg protein was loaded for gel electrophoresis and immunoblotting. For alkaline phosphatase  treatment, 20 μg protein was diluted in 50 mM Tris-HCl pH 8.5 and incubated at 37°C for 1 hr prior to gel loading.Yeast strains were cultured and processed as previously described  with hyp2HPO4, 2 mM KH2PO4, 0.05%(v/v) Tween 20, pH 7.4) then incubated in primary antibody followed by an appropriate secondary (IRDye800CW or IRDye680RD mouse or rabbit IgG) and scanned using LI-COR Odyssey CLx imaging system. For immunopurification samples, IRDye 680RD Detection Reagent was added to primary antibody incubation.Immunoblotting was performed as described in 6 cells/ml and 3 μl were spotted on plates containing rich medium and rich medium supplemented with 1.6 mM CoCl2. Plates were incubated at 30°C for the indicated time. For random spore analysis, strains were mated for two days on malt-extract plates at room temperature prior to glusulase treatment (0.5% [v/v]) for 16 hr. Spores were then diluted to 1.6 × 106 cells/ml and spotted on rich medium or rich medium plus nourseothricin (100 µg/ml) and G418 (100 µg/ml).Strains grown on rich medium agar for two days were diluted in water to 1.6 × 10S. pombe and RT-qPCR analysis have been described previously (23VN primers (NEB). The tested genes were quantified by real-time PCR using SYBR Green qPCR master mix (Promega). tub1+ served as the internal control to calculate the relative expression across different samples. Error bars represent ± SEM of fold changes from three biological replicates.Total RNA preparation from eviously . BrieflyRPS23 cDNA was cloned into the pProEX HTb bacterial expression vector (Invitrogen) and protein expression was induced in E. coli BL21-CodonPlus (DE3)-RIPL competent cells (Agilent) with 0.6 mM IPTG at 37°C for 2 hr in LB medium. Cells were pelleted and solubilized in lysis buffer   at room temperature for 2 hr. Lysates were cleared by centrifugation in a JA20 rotor (Beckman Coulter) at 16,000 rpm for 40 min and incubated with Ni-NTA agarose  on a rotator for 2 hr at room temperature. Lysates plus resin were transferred to columns and washed 3x with lysis buffer. 6xHis-RPS23 was eluted 3x with lysis buffer adjusted to pH 3.5 and dialyzed overnight into 25 mM phosphate pH 6.8, 0.25 M NaCl, 2 mM DTT, and 1 mM PMSF.Human Homo sapiens using the Mascot V2.2.6 (Matrix Science) search engine running through Proteome Discoverer v1.3 (Thermo Scientific) with fragment match tolerance set to 0.03 Da.In vitro hydroxylation reaction with Ofd1 and 6xHis-RPS23 was digested with 1 μg trypsin (0.2 μg/μl in 50 mM acetic acid) for 2 hr at 37°C and quenched with the addition of TFA to a final concentration of 0.1% [v/v]. Peptides were analyzed using LTQ Orbitrap Velos MS (Thermo Fisher Scientific) and parent masses (393.22 Da and 401.21 Da) were selected by SIM scan. Peptides were searched against the RefSeq 2012 database  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Randy Schekman as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Coupled prolyl dihydroxylation and nuclear import of pre-ribosomal uS12/Rps23 regulate fungal hypoxic adaptation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This paper describes the role of prolyl dihydroxylation of the small ribosomal subunit protein Rps23 in regulation of sterol synthesis pathways in fission yeast. It builds on a substantial body of work by the senior author on the elucidation of this pathway, in particular the identification of Ofd1 as a 2-oxoglutarate-dependent oxygenase that generates an oxygen-dependent signal leading indirectly to the degradation of the key transcription factor Sre1N.In outline, it is now proposed that hydroxylation of Rps23 is promoted by the assembly of a protein complex involving (at least) Ofd1, Rps23 and a previously identified Ofd1-interacting protein Nro1. Binding of Ofd1 to the Nro1/Rsp23 complex is proposed to be competitive with Sre1N and modulated by the Ofd1-dependent hydroxylation of Rps23. Since binding of Ofd1 to Nro1 prevents the interaction with (and degradation of) Ser1N, production of Rsp23 and the catalytic rate for hydroxylation confer regulation on the sterol response (including by oxygen availability). This action that is proposed to be important in the co-ordination of biosynthetic processes.The reviewers agreed that the work is of high quality and in general supports the conclusions. The manuscript should also be of interest to the wider biomedical community since Ofd1 is conserved in man as OGFOD1 and RPS23 has been identified as a prolyl hydroxylated substrate of human OGFOD1, raising questions as to the nature of signaling in human cells.eLife but the author will need to address the following points prior to publication.Based on this there was a consensus that the work is in principle appropriate for Essential revisions:1) To strengthen Rps23 specificity in hypoxia, additional controls are suggested. In IPs with cross-linker  an addit2) Could the authors comment on 3) The authors demonstrate the crucial residues involved in interaction between Ofd1 and Rps23/Nro1 in vitro . However4) The authors implicate a tight coupling between dihydroxylation of Rps23 and nuclear import . What ha5) Does Nro1 directly import Rps23 into the nucleus? Or does it serve as an adaptor for other import receptors? Given that Nro1 is not essential for yeast cell viability, but Rps23 is, it is important to better dissect the import pathway(s) of Rps23. 1) To strengthen Rps23 specificity in hypoxia, additional controls are suggested. In IPs with cross-linker  an additWe agree that Rps5 is an important negative control. In response to the reviewers’ suggestion, the IPs in 2) Could the authors comment on rps23Δ and rps2302Δ cells. The Ofd1-Rps23 interaction is difficult to detect in the absence of crosslinker  The authors demonstrate the crucial residues involved in interaction between Ofd1 and Rps23/Nro1 in vitro . Howeverrps2302Δ sre1N and nro1Δ sre1N cells when expressed from a plasmid. If the Ofd1-binding sites in Nro1 and Rps23 contribute equally to the sequestration of Ofd1, our model predicts that the Ofd1-binding mutants will not restore growth on cobalt.As suggested by the reviewers, we tested if the Ofd1-binding sequences in Nro1 and Rps23 are required to sequester Ofd1 in vivonew . We mutaIn the case of Nro1, the residues that blocked Ofd1 binding in vitro also failed to rescue growth on cobalt, consistent with the model. We verified that expression of the Nro1 mutants was similar to wild-type and directly tested Sre1N activation under +/- oxygen conditions . As expe4) The authors implicate a tight coupling between dihydroxylation of Rps23 and nuclear import . What haofd1Δ and ofd1 H142A D144A backgrounds. Furthermore, we imaged rps2302 P62A-GFP cells. In these strains, Rps23 is not hydroxylated and localizes to the nucleus. Thus, import of Rps23 does not require P62 hydroxylation which is expected given the viability of ofd1Δ and ofd1 H142A D144A cells. In addition, To address this concern, we expanded 5) Does Nro1 directly import Rps23 into the nucleus? Or does it serve as an adaptor for other import receptors? Given that Nro1 is not essential for yeast cell viability, but Rps23 is, it is important to better dissect the import pathway(s) of Rps23.nro1Δ cells, the Nro1-dependent pathway cannot be the only route for nuclear import of Rps23. Further, it remains to be tested whether Nro1 is sufficient to import Rps23. As the reviewers note, Nro1 may act as an adaptor that links Rps23 to an importin. Previous studies published by our lab identified the importin-β Kap123 as an Nro1-binding partner, and both Nro1 and Ofd1 are mislocalized in kap123Δ cells . These data suggest that Kap123 may be an additional member of the complex. However, rps23Δ kap123Δ double mutants, but not rps23Δ nro1Δ cells, show severe growth defects, suggesting that Kap123 may separately function in Rps23 import.This is an excellent point, and we agree that this is an interesting area for future study. Given the viability of