A lower synthesis temperature resulted in polycrystalline Mn-doped ZHN. Under isochronal annealing up to 250 °C the bulk ZHN and the minority phases from the ZnO samples decomposed into ZnO. The Mn2+ ions distribution in the annealed nanocrystals was significantly altered, varying from a uniform volume distribution to a preferential localization in the outer layers of the nanocrystals. Our results provide a synthesis strategy for tailoring the dopant distribution in ZnO nanocrystals for applications ranging from surface based to ones involving core properties.The synthesis of semiconductor nanocrystals with controlled doping is highly challenging, as often a significant part of the doping ions are found segregated at nanocrystals surface, even forming secondary phases, rather than incorporated in the core. We have investigated the dopant distribution dynamics under slight changes in the preparation procedure of nanocrystalline ZnO doped with manganese in low concentration by electron paramagnetic resonance spectroscopy, paying attention to the formation of transient secondary phases and their transformation into doped ZnO. The acidification of the starting solution in the co-precipitation synthesis from nitrate precursors lead to the decrease of the Mn Whether the doping ions are incorporated or remain outside the nanocrystals core, they influence the crystal growth, initiating and controlling morphological and even structural changes15. As previously shown, sometimes only a small fraction of dopant ions is actually present inside the nanocrystals, the largest part being localized in the intergrain region, often in secondary phases18. In many cases, at lower dopant concentration levels (below 1 at%), these secondary phases are found as a thin amorphous shell containing isolated dopant ions19. For larger dopant concentrations impurity-rich secondary phases are formed which affect the magnetic, electrical and optical properties of the nanostructured semiconductors26. The amount and composition of the material in the intergrain region is strongly dependent on the synthesis conditions, which opens interesting possibilities to tailor the semiconductor nanostructures for applications requiring special surface properties  or different optical, electrical and/or magnetic characteristics29. In order to achieve this level of control we would also need to strengthen our understanding of the doping mechanism at nanoscale, especially of the influence of the synthesis parameters and precursors on the dopant localization and doping efficiency.However, doping colloidal semiconductor nanocrystals is still an experimental challenge33 and/or electron tomography36 provide spectacular results in imaging the local distribution of chemical elements. More recently, atom probe tomography (APT) boosted the knowledge in the field of the chemical elements distribution and segregation phenomena at atomic scale38. Still, all these state-of-the-art techniques have a strong local character, being less statistically relevant and not always suitable for dopant concentrations lower than a few percent.The ability to determine the spatial distribution of low concentrations (below 1 at%) of doping ions in nanostructured materials, as well as the accuracy of these determinations, are yet another experimental challenge. Advanced techniques of chemical mapping at nanometric scale or even atomic spatial resolution by aberration-corrected STEM (scanning transmission electron microscopy) and EELS (electron energy loss spectroscopy) mappingparamagnetic dopants present in low concentrations in nanostructured semiconductors46.Alternatively, electron paramagnetic resonance (EPR) spectroscopy offers a sensitive and statistically reliable method to determine the distribution/localization of 2+ ions (nano-ZnO:Mn) prepared by co-precipitation from nitrate precursors followed by mild thermal treatments . We aim to demonstrate a simple and cost-effective method to tailor the dopant distribution of the ZnO nanocrystals for specific applications. An important part of this approach refers to the control of the amount and chemical composition of the secondary intermediate phases, expected to contain a non-negligible concentration of dopant Mn2+ ions, by minor adjustments of the synthesis route. These secondary phases were analysed by EPR and Fourier transform infrared (FTIR) spectroscopies combined with microstructural investigations. The dynamics of the dopant distribution under mild isochronal thermal treatments was monitored by EPR. By manipulating the secondary phases, we were able to obtain ZnO nanocrystals with tailored dopant distributions, from uniform to dopant-rich surfaces.The present work is focused on the assessment and control of the dopant distribution in nanocrystalline ZnO doped with a low concentration (0.1 at%) of MnIn order to assess the influence of the synthesis parameters on the dopant distribution and the formation of intermediate secondary phases, we have prepared several samples following a similar synthesis route, but varying one synthesis parameter from sample to sample.3mc, JCPDS 89–1397) with lattice parameters a = 3.254(3) Å, c = 5.214(5) Å. The calculated average crystallite size varies with the amount of nitric acid added during the synthesis from 38 ± 3 nm in the sample called ZOM1 to 25 ± 2 nm in the sample called ZOM2, corresponding to the smallest (50 mL) and largest (250 mL) amount of nitric acid, respectively. The XRD patterns of these two samples, selected for further spectroscopic and microstructural investigations, are presented in Fig. First, we have prepared a series of five nano-ZnO:Mn samples using different amounts of nitric acid to acidify the starting solution. These samples exhibit similar XRD patterns, indexed as single phase ZnO - wurtzite 8(NO3)2·2H2O – ZHN  with a calculated average crystallite size higher than 100 nm. This sample was labelled ZHNMn. Similar results concerning different reaction products associated with the modification of the synthesis temperature have been reported in ref.47.The second synthesis parameter that we changed was the synthesis temperature. Another sample was prepared by following the same synthesis steps as for ZOM2, but lowering the synthesis temperature from 80 °C to 35 °C. The XRD pattern of this sample Fig. , bottom 51, there are several possible thermal decomposition paths of bulk ZHN into ZnO, all of them at temperatures below 200 °C. All reports point to a sequential process involving different intermediary compounds and mechanisms, depending on the synthesis conditions and post-synthesis treatments, expected to affect also the dopant distribution. The thermal decomposition of the ZHNMn sample to ZnO was monitored by XRD and is represented in Fig. 3(OH)4(NO3)2 and 10% Zn5(OH)8(NO3)2·2H2O. After further isochronal annealing up to 170 °C, two crystalline phases in almost equal quantities are observed, namely Zn3(OH)4(NO3)2 and ZnO. Finally, after isochronal annealing up to 200 °C the decomposition of the ZHNMn sample into nano-ZnO  is complete. Only a barely detectable amount (about 1%) of ZnOHNO3·H2O with poor crystallinity was identified 8(NO3)2·2H2O in ref.49.According to literature3mc. The particle size distribution for the ZOM1 sample  images of ZOM1 Fig.  and ZOM22 range. The SAED pattern 8(NO3)2·2H2O crystallized in monoclinic structure with C2/m space group. It is important to emphasize here that observing the ZHNMn sample in the as-prepared status was possible only by working in low-beam conditions. The increase of the electron irradiation dose, like for working in HRTEM conditions, lead to the full transformation of ZHNMn into ZnO:Mn.The TEM investigation of the ZHNMn sample Fig.  has reveern Fig.  recorded3·H2O minority phase observed in the XRD pattern of this sample 8(NO3)2·2H2O compound58. The sharp peak around 3570 cm−1 and the strong peak at 3400 cm−1 were assigned to various stretching vibrations of the O-H bonds in Zn5(OH)8(NO3)2·2H2O. The broad band around 3300 cm−1 and the peak around 1640 cm−1 indicate the presence of water molecules in the interlayer space of this layered hydroxide salt or adsorbed on the surface. In the spectral range 1500-800 cm−1 there are several well defined absorption bands characteristic to the nitrate group. Thus, the most intense peak around 1380 cm−1 corresponds to the ν3 vibration (NO2 stretching mode of a free nitrate ion), while the weak peaks around 1050 cm−1, 840 cm−1 and 720 cm−1 correspond to the ν1 (NO stretch of a free nitrate group), ν2 (out-of-plane vibration) and ν4 (NO2 bend) vibrations, respectively. The presence of only a very weak and broad band at 1050 cm−1, as well the fact that the absorption band at around 1380 cm−1 is not split, indicate that the nitrate ions are not coordinated to the central Zn ions in the ZHNMn sample. This is indeed expected for the layered structure of Zn5(OH)8(NO3)2·2H2O55. The weak shoulder at ~1500 cm−1 was associated to the partial dehydration of Zn5(OH)8(NO3)2·2H2O55. This assignment applies to the 1500 cm−1 band observed in the ZOM2 spectrum too.The FTIR spectrum of the ZHNMn sample is similar with the reported infrared spectra of the Zn−1 is almost absent, showing that the crystallization water molecules were removed, while the weak band at 1640 cm−1 proves that the surface water is still present. The weak peak at 1500 cm−1 was assigned to the asymmetric stretching vibration mode for the nitrate ion in zinc hydroxynitrate with the structural formula Zn3(OH)4(NO3)2, where the nitrate ion is coordinated directly to the metallic centre55. The small shoulder observed around 1330 cm−1 is also assigned to the coordinated nitrate, in agreement with the structure of Zn3(OH)4(NO3)2. The medium absorption band observed around 460 cm−1 is assigned to the Zn-O bond in ZnO52. These results, in good agreement with the XRD data, indicate that in the sample annealed at 130 °C three phases co-exist: Zn5(OH)8(NO3)2·2H2O, Zn3(OH)4(NO3)2 and ZnO. The annealing of the ZHNMn sample at 170 °C resulted in an increase in the intensity of the absorption bands of the nitrate ion around 1500 cm−1 and 1050 cm−1. These changes are in agreement with the formation of the Zn3(OH)4(NO3)2 as a major phase, along with the ZnO phase, as observed from the XRD analysis. After annealing at 200 °C, FTIR indicates the presence of ZnO as a predominant phase, as well as the presence of yet another secondary phase containing hydroxide and nitrate groups, which according to the XRD data corresponds to the minority phase ZnOHNO3·H2O. This is supported by the splitting of the 1380 cm−1 band (ν3 vibration mode of the nitrate ion) observed in the FTIR spectra of the sample annealed at 170 and 200 °C55. The FTIR spectrum of the sample annealed at 240 °C contains an absorption maximum around 450 cm−1, characteristic to the ZnO predominant phase with an isotropic morphology of the nanocrystals. Also, around 1380 cm−1 an intense narrow peak is observed, characteristic to the free nitrate ion. However, given the shape and intensity of this band, it is more likely that it belongs to NO2 gas molecules captured on the surface of the ZnO nanocrystals, as a result of the decomposition of the zinc hydroxynitrates50. This narrow peak is also observed with lower intensity, superimposed on a broader peak in all as-prepared samples  localized in different host lattices (see Supplementary Information).The EPR spectra of the as-prepared samples are presented in Fig. 2+ ions substitutionally localized in Zn2+ sites in ZnO59. This complex sextet is generated by two manganese centres, called Mn2+(c) and Mn2+(d), consisting of Mn2+ ions localized in ZnO nanocrystals and disordered ZnO, respectively , corresponding to Mn2+ ions localized in a host lattice different from ZnO that we denoted “phase x”. The spin Hamiltonian (SH) parameters of the three Mn2+ centres are listed in Supplementary Information Table In the case of the ZOM1 sample, the EPR spectrum is dominated by six groups of close lying lines with a complex pattern, with a ~7.8 mT separation between the groups   and Mn2+(d) in ZnO and Mn2+(y) in a disordered “phase y”, with SH parameters listed in Table The EPR spectrum of the ZOM2 sample resembles the spectrum of the ZOM1 sample, although the intensities of the EPR lines attributed to the Mn(y) Fig. . The bes(y) Fig.  has been2+ paramagnetic centres in the ZOM2 sample shows that only ~21% of the Mn2+ ions are localized in ZnO  centre. As observed in the TEM image of the as-prepared ZOM2 sample  centres is expected to be high enough to determine increased magnetic dipolar interactions between the Mn2+ ions, which would explain the broad EPR lines of the Mn2+(y) spectrum.The analysis of the Mnple Fig. , the ZnO2+(y) centres should be similar with the EPR parameters of the Mn2+ ions in ZHNMn. From structural investigations two cationic sites were identified in Zn5(OH)8(NO3)2·2H2O, one with the Zn2+ ions octahedrally coordinated by six OH− groups and one with Zn2+ ions tetrahedrally coordinated by three OH− groups and one water molecule60. Figure 2+(I) and Mn2+(II), were identified. The SH parameters determined for the Mn2+(I) centres  centres indicate a local crystal field with axial symmetry, which could correspond to Mn2+ ions localized in the tetrahedrally coordinated cationic sites. To the best of our knowledge, this is the first report of the EPR parameters of Mn2+ in ZHN. These parameters can be further used as reference data, especially as there is a growing scientific interest in the investigation of brucite-like layered hydroxide salts (including ZHN) due to both ion-exchange property and intercalation capacity63.As the synthesis procedure is similar for the ZOM2 and ZHNMn samples, except for the synthesis temperature, we would assume that the y phase is related to ZHN. Moreover, as shown in Fig. 2+(y) centres in the ZOM2 sample are close to the parameters of the two Mn2+ centres in ZHNMn. The Mn2+(y) spectrum could be well reproduced by a summation of the spectra calculated with the Mn2+(I) and Mn2+(II) parameters, considering a disordered host lattice (see Supplementary Information) and broad lines (ΔB = 1.0 mT). This result supports the assignation of the Mn2+(y) spectrum to Mn2+ ions localized in a disordered Zn5(OH)8(NO3)2·2H2O shell surrounding the ZnO nanocrystals.As seen in Table 2+ ions in the as-prepared samples gradually transforms into the characteristic spectrum of Mn2+ ions in ZnO, following the annealing induced transformation of the host lattice into ZnO. We have selected from Fig. 2+ centres and follow their transformation under annealing in the two samples.Further information was obtained by comparing the behaviour under annealing of the ZHNMn and ZOM2 samples, as reflected in the evolution of the corresponding EPR spectra versus the isochronal annealing temperature. As presented in Fig. 2+(I) and Mn2+(II) EPR spectra become weaker, disappearing completely at 120 °C  and Mn2+(d) centres in ZnO , exhibit very narrow lines with a larger line separation (~9.4 mT), and are visible in the EPR spectra of the annealed sample up to 170 °C. These centres could correspond to Mn2+ ions localized substitutionally in Zn2+ cationic sites in the intermediate Zn3(OH)4(NO3)2 phase, identified by XRD. In this compound the cationic sites are sixfold coordinated by four OH− and two NO3− groups, which are expected to create a local crystal field with higher anisotropy than in the case of the Mn2+(I) centres coordinated by six OH− 63.By annealing the ZHNMn sample at temperatures above 100 °C the Mn °C Figs  and 6a. 2+ ions are localized in ZnO 8(NO3)2·2H2O into ZnO is initiated at lower temperatures in the sample regions containing manganese than in the pure ones. The EPR spectrum of the ZHNMn sample annealed at 200 °C contains only the lines of the Mn2+(c) centres, showing that all Mn2+ ions are now localized in the core of the ZnO nanocrystals.After the 170 °C annealing step all MnZnO Fig. . This re2+(c) centre in ZnO is correlated with the crystallite size, following an empirical law64. According to this relationship, the crystallite size of the ZnO nanocrystals formed by the thermal decomposition of the ZHNMn sample at 200 °C would be around 52 nm (see Supplementary Information for more details). This value perfectly matches the crystallite mean size resulted from the XRD analysis of this sample. This is a very important result, proving the uniform distribution of the Mn2+ ions inside the ZnO nanocrystals resulted from the thermal decomposition of ZHNMn.The EPR broadening parameter of the Mn2+(y) centres into both Mn2+(c) and Mn2+(d) centres proceeds at a slower pace, their spectrum being still visible at 250 °C 8(NO3)2·2H2O phase hosting the Mn2+(y) centres takes place at higher temperatures than for the ZHNMn sample, without the formation of intermediate compounds. We assume that this behaviour is due to the size (a few atomic layers) and disordered state of the ZHNMn shell surrounding the ZnO nanocrystals. Such a size induced disparity in the thermal behaviour was previously observed in the case of the disordered nanometric ε-Zn(OH)2 shell on ZnS nanocrystals, which decomposed into ZnO at higher temperatures and in different steps than polycrystalline ε-Zn(OH)219.In the case of the ZOM2 sample, the thermally induced transformation of the Mn °C Figs b and 6b.2+(c) centres spectrum in ZOM2 which, according to ref.64, would correspond to a crystallite size of ~25 nm, does not vary across the whole annealing range. In fact, the Mn2+(c) spectrum does not change with annealing, except for increasing in intensity. After the final annealing step at 250 °C the distribution of the Mn2+ ions in ZOM2 changed to 81% localized inside the ZnO lattice (centres Mn2+ (c) and Mn2+ (d)), and 19% embedded in the remaining ZHNMn (Table The broadening parameter of the Mn2+(x) lines at 170 °C due to the thermal decomposition of the x phase into ZnO. Indeed, after the final annealing step at 200 °C the Mn2+ ions are all localized inside ZnO (Table 2+(c) spectrum did not change after annealing. Given that the amount of Mn2+(d) centres remained practically the same after annealing and that the crystallization process of ZnO necessitates higher temperatures/annealing times65, we assume that the Mn-rich ZnO resulting from the decomposition of the x phase was added directly as crystallized layers on the existing ZnO nanocrystals.The EPR spectra of the ZOM1 sample submitted to isochronal thermal annealing up to 200 °C .The as prepared ZOM1 sample has 44% of the dopant ions uniformly distributed in the ZnO nanocrystals core, 19% are localized in the disordered ZnO phase, while 37% are found in rests of precursors. The thermal annealing up to 200 °C affects only to a very small extent the sample structure, as the secondary phase detected in the as-prepared sample is in a very small amount. The thermally induced decomposition of this phase into ZnO leads to a quite interesting distribution of the Mn2+ ions are added to the outer layers of the ZnO nanocrystals, while some of the nanocrystals are still covered by a mixture of disordered ZnO and ZHN. These results are supported by the structural information from the TEM-HRTEM images of the ZOM2 sample after annealing at 250 °C. Figure On the other hand, for the as-prepared ZOM2 sample, consisting of smaller size ZnO porous nanocrystals, most of the dopant ions (79%) are localized in a minority phase of amorphous ZHN surrounding the ZnO nanocrystals and only 17% of them are found in the ZnO core, according to EPR. After the last annealing step at 250 °C, 41% of the Mn2+ ions uniformly distributed inside the ZnO lattice. The relatively low temperature (200 °C) at which the uniformly doped ZnO nanocrystals are formed by this synthesis route is interesting for applications, given that the growth of ZnO nanocrystals of such size from a pure disordered ZnO phase would need temperatures above 500 °C64. Such nanocrystals could be interesting for applications relying on volume properties, like optoelectronics. ZnO nanocrystals with uniform Mn2+ distribution were previously obtained from the thermal decomposition of hydrozincite, but at higher temperatures67. Our results show that the thermal decomposition of doped precursors at low temperatures (below 300 °C) is a reliable, simple and cost-effective method for the preparation of uniformly doped nano-ZnO:Mn.The 200 °C annealed ZHNMn sample consists of ZnO nanocrystals with better crystallinity Fig. , with al2+ ions in low concentration prepared by co-precipitation. By varying one synthesis parameter from one sample to another we obtained two nano-ZnO:Mn samples with different dopant distributions and minority phases and a polycrystalline Mn-doped zinc hydroxynitrate sample (ZHNMn). The minority phases from the two nano-ZnO:Mn samples were identified as residues of precursor nitrate compounds in the ZOM1 sample and ZHN in the ZOM2 sample.In summary, we have investigated the effect of slight changes in the synthesis procedure on the dopant distribution in nanocrystalline ZnO doped with Mn2+ ions were substitutionally localized in the two types of Zn2+ sites. The isochronal annealing treatment up to 200 °C lead to the complete transformation of the ZHNMn sample into nanocrystalline ZnO, with all Mn2+ ions uniformly distributed in the core of the ZnO nanocrystals.In the as-prepared ZHNMn sample the Mn2+ ions were incorporated in ZnO, while 79% of them were localized in the minority ZHN phase, present as a thin amorphous layer around the ZnO nanocrystals. This is already an interesting result, showing the possibility to functionalize the ZnO nanocrystals with ZHN, a layered material with various applications47. The thermal annealing process up to 250 °C lead to the decomposition of three quarters of ZHN directly into ZnO, with part of it adding as crystalline layers on the initial nanocrystals and the rest remaining as disordered ZnO. After the last annealing step the ZnO nanocrystals contained 58% of the Mn2+ ions, 17% uniformly distributed in the core and 41% in the outer layers.A very different situation was encountered for the as-prepared ZOM2 sample: only 21% of the Mn2+ ions in the annealed ZOM1sample is an intermediate case, with a smaller difference between the core and surface concentrations than in the ZOM2 sample.The distribution pattern of the MnOur results demonstrate a strategy to tailor the dopant distribution in colloidal ZnO nanocrystals by small adjustments of the preparation algorithm by a simple and cost-effective method. Thus, the most appropriate route to obtain a uniform dopant distribution in the ZnO nanocrystals volume seems to be the thermal decomposition of a doped precursor, while a non-uniform distribution with dopant-rich layers at the nanocrystals surface can be obtained from the thermal decomposition of minority phases. ZnO nanocrystals covered by a dopant-rich shell are of interest for surface sensitive applications like catalysis or gas-sensing, while uniformly doped ZnO:Mn nanocrystals are suitable for applications in nanoelectronics, optoelectronics or spintronics. These findings are expected to contribute to a better understanding of the doping mechanisms at nanoscale and to increase the applicative opportunities.2+ ions were prepared by co-precipitation using the same precursors and steps for all samples. An aqueous solution containing zinc nitrate (0.19 M) and manganese nitrate (0.02 M), corresponding to 0.1 at% Mn2+ nominal concentration, has been prepared at 80 °C and subsequently acidified with HNO3 1 M and magnetically stirred for several minutes. The amounts of HNO3 1 M added to the different samples were in the range of 50 to 250 mL in steps of 50 mL. Afterwards, this mixture was precipitated with a strong basic solution of NaOH 2.4 M (in excess) added slowly with a peristaltic pump, under continuous magnetic stirring. The pH values at the end of the syntheses varied from 10 to 5 for the corresponding 50 to 250 mL HNO3 1 M additions. The suspension thus obtained was left to age for one hour at the same temperature, under continuous magnetic stirring. The precipitate was collected by centrifugation and washed several times with bi-distilled water and absolute ethanol, then air dried at 60 °C.Several samples of nano-ZnO doped with 0.1 at% nominal concentration of Mn3 1 M of 250 mL, but lowering the synthesis temperature to 35 °C. The XRD investigation identified this sample as zinc hydroxynitrate [Zn5(OH)8(NO3)2·2H2O].Another sample was prepared following the same procedure with the maximum amount of HNOλ = 1.54184 Å), in the θ − θ geometry. The lattice parameters of the ZnO crystalline phases and the average crystallites size were determined with the Topas v.3 software (Bruker) by Rietveld refinement.XRD investigations were performed with a Bruker D8 Advance X-ray diffractometer provided with a Cu anode and Ni filter  modes in order to gain information regarding the shape and size of the nanograins as well as the likely presence of secondary phases. The ZHNMn sample was investigated in low-beam conditions to avoid the amorphization or transformation into ZnO.−1 spectral range, by accumulating 128 scans at a resolution of 4 cm−1. The powdered samples were diluted into KBr powder in a 1:100 mass ratio, ground thoroughly and pressed into pellets.FTIR spectra were recorded with a Spectrum BX II (Perkin Elmer) spectrometer in the 4000-350 cm-band (34 GHz) EPR measurements were performed at room temperature (RT) with the Bruker E500Q spectrometer from the Centre for advanced ESR/EPR techniques (CetRESav - http://cetresav.infim.ro/). The spin Hamiltonian (SH) parameters of the paramagnetic centres were determined with the EASYSPIN v.5.1.11 software68.EPR investigations were performed on powder samples inserted in calibrated pure fused-silica tubes of 2 mm inner diameter. QIsochronal annealing experiments from 100 °C up to 250 °C were performed in air in 10 °C steps in order to study the thermal evolution of the samples. The sample was annealed at each set temperature in a temperature stabilized (±1 °C) furnace for 10 min. and then cooled down to RT for complementary measurements by XRD, EPR and FTIR.Supplementary Information


Less well understood is the extent to which aerenchyma permits venting of respiratory carbon dioxide (CO2) in the opposite direction. Large, potentially toxic concentrations of dissolved CO2 develop in submerged rice soils. We show using X‐ray computed tomography and image‐based mathematical modelling that CO2 venting through rice roots is far greater than thought hitherto. We found rates of venting equivalent to a third of the daily CO2 fixation in photosynthesis. Without this venting through the roots, the concentrations of CO2 and associated bicarbonate (HCO3−) in root cells would have been well above levels known to be toxic to roots. Removal of CO2 and hence carbonic acid (H2CO3) from the soil was sufficient to increase the pH in the rhizosphere close to the roots by 0.7 units, which is sufficient to solubilize or immobilize various nutrients and toxicants. A sensitivity analysis of the model showed that such changes are expected for a wide range of plant and soil conditions.The growth of rice in submerged soils depends on its ability to form continuous gas channels—aerenchyma—through which oxygen (O 2 accumulate in submerged paddy soils because CO2 from plant and soil respiration escapes only very slowly. We found, using X‐ray computed tomography and image‐based mathematical modelling, venting of soil CO2 through rice roots at rates equivalent to a third of the daily CO2 fixation in photosynthesis. Without this venting, the concentrations of CO2 and associated bicarbonate in root cells would have been well above levels known to be toxic to roots. Removal of soil CO2 and hence carbonic acid will also affect the solubility and hence plant uptake of various nutrients and toxicants in the rhizosphere.Large, potentially toxic concentrations of dissolved CO A key problem is how to separate the fluxes of soil‐derived CO2 by volume and 60% CH4. We developed a mathematical model to account for these observations on the basis of the following picture of events.In brief, we grew initially 4‐week‐old rice seedlings in a submerged, anaerobic rice soil contained in glass pots, and, after 4 weeks, scanned the pots using X‐ray CT imaging to measure the spatial distribution of roots and gas bubbles entrapped in the soil Figure c. The im2 or CH4, or other volatile products of respiration, gas bubbles will form and tend to become entrapped beneath soil particles. If the bubbles become sufficiently large, or if the soil is agitated by some mechanical disturbance, then the bubbles will rise to the surface by “ebullition.” At steady state .We describe the steady‐state transport of each dissolved gas through the soil by the following continuity equation:Di = DiLθLfL where DiL is the diffusion coefficient in free solution, θL is the soil volumetric water content, and fL is a tortuosity factor  is proportional to the sum of the partial pressures of the volatile solutes, ∑Pi=PCO2+PCH4+PN2+PH2O (PH2O is the saturating pressure of H2O):Kθ is a constant that is characteristic of the submerged, puddled soil. From the gas law: CiG is the concentration of gas i in the soil gases. From Henry's law: KiH is the dimensionless Henry's law constant for gas i.In Equation or Kirk, . The volSi, Ei, and Ri. For Si, at steady state, CO2 production from soil carbon is constant with depth and time, equal to SCO2,0, and production from root‐derived carbon is proportional to the root length density, LV (root length per unit soil volume), that is,kV is a proportionality constant. At steady state, the ratio of CH4 production to CO2 production is also constant SCH4Ei, the rate of ebullition is a function of the volume of the gas bubbles: As bubbles grow, they become more buoyant and so are more easily displaced. Hence, taking total gas volume to represent bubble volume:kE is a rate constant that depends on the physical properties of the soil. For Ri, root‐mediated efflux from the soil occurs by degassing of dissolved CO2 and CH4 into the root aerenchyma and diffusion through the aerenchyma to the atmosphere  8.5, CEC 9.0 cmolc kg−1, organic carbon content 73 g kg−1, and carbonate content 96 g kg−1 . Portions of topsoil (0‐ to 30‐cm depth) were air dried and sieved to pass <2 mm. The properties of the sieved soil were 42% clay, 40% silt, pH  Perspex pots to a depth of 17 cm. The resulting soil bulk density was 0.81 kg dm−3, and the volumetric water content was 0.69. The filled pots were inserted into 12‐cm‐diameter, 21‐cm‐deep glass pots, and the space between the inner and outer pots were filled with further slurry. This arrangement ensured anoxic conditions in the soil in the inner pot, whereas the thin Perspex wall of the pot was completely transparent to X‐rays for imaging after removal from the outer pot. Further deionized water was added to bring the level to the top of the pots, and the water standing in the pots was maintained at this level through the experiment. The soil was allowed to become reduced for 4 weeks at 30°C before transplanting the rice seedlings.Portions (1.2 kg) of the air‐dried soil were mixed with 10 g kg−2·s−1 white light) at 30°C and 10.5‐hr dark at 24°C.Rice seeds (CV IR55179) were germinated in petri dishes at 30°C in complete darkness for 3 days. The germinated seeds were transferred to a mesh floating on Zn‐free Yoshida nutrient solution  and pH . Redox potential was monitored with a Pt electrode. The composition of gas bubbles accumulated in the soil was monitored by periodically fitting over each pot a 3‐dm3 gas‐tight bag fitted with a sampling port and agitating the pots to displace entrapped soil gases into the headspace. Samples of the headspace were withdrawn by syringe and analysed for CO2 and CH4 by gas chromatography (Cambridge Scientific Instruments 200 Series GC).Further pots were set up in the same way but left unplanted to measure gas productions in the bulk soil following flooding. Each pot was fitted with a rhizon solution sampler  with a 5‐cm porous section and fitted with a Luer lock. The samplers were held vertically in the soil so that the porous section ran from 8.5 to 13.5 cm below the floodwater–soil boundary. At weekly intervals, solution was withdrawn and analysed for dissolved CObHS) of the submerged, reduced soil from the results of Affholder et al.  concentration, the addition of H+ in Fe (II) oxidation by the roots was far smaller. Hence, bHS = (11.0 − 1.6)/0.32 = 29 mmol·kg−1·pH−1.We estimate the pH buffer power . Data were reconstructed using a filtered back‐projection algorithm implemented in Nikon CTPro 3D, generating 32‐bit volumes that were subsampled to produce a stack of two‐dimensional eight‐bit Tagged Image File Format files for each scan. A modest beam hardening correction was applied during reconstruction.Gas bubbles were extracted from the data by 3D median filtering using an 8 × 8 × 8 voxel cubic kernel, then hysteresis thresholding, using the Fiji image analysis software  using the conversion that one voxel edge length was equivalent to 0.04 mm. Each scan was 5.8 cm  in depth, with approximately 5 cm of soil above the upper edge and 6 cm below the lower edge. The LV and θG data were extrapolated over the entire depth by fitting three‐dimensional Gaussian distributions to the pooled data for the three replicates for each planting density:X is either LV or θG and φ, σr, and σz are the corresponding fitting coefficients. Parameters were fitted in MATLAB using the fmincon function to minimize the square difference between the measurements and Equation We transformed the scanned root and gas data into volumetric spatial data  Kθ,SCO2,0, and αCH4 in Equations 2 and pH in the unplanted bulk soil and the ratio of CO2 to CH4 measured in entrapped gases displaced from the soil; and (c) all other variables, except kE, kT, and kV, based on the experimental data and standard values for the constants and coefficients  the three‐dimensional distribution of kE, kT, and kV by running the model to obtain the best agreement between our observed and predicted three‐dimensional profiles of θG for each planting density, using the MATLAB fmincon function. A unique set of kE, kT, and kV values was found for the whole data set by minimizing the average of the fitting errors calculated for the individual replicate runs.We then fitted values of 2 in the soil per unit soil surface was calculated fromThe rate of generation of COL and R are the depth and radius of the soil volume, respectively. The flux through the roots was calculated fromwhere The flux from the soil surface by ebullition was calculated fromThe flux from the soil surface by diffusion was calculated fromhttps://doi.org/10.17862/cranfield.rd.7628870.Copies of the experimental data and the source code for the model written in FORTRAN are available from 33.1kV, kE, and kT values  = 0.016 mol day−1 and the flux of CO2 through the roots per pot (=JR × soil surface area) = 0.005 mol day−1. The plant shoot growth over 28 days was 2.2 ± 0.4 g dry weight per pot ≈ 0.073 mol C. Assuming exponential growth and equal root and shoot growth, this is equivalent to approximately 0.015 mol day−1 after 28 days. So the CO2 flux though the roots was approximately a third of the daily rate of photosynthesis. For the standard values, the proportions of total CO2 escaping though the roots and by diffusion and ebullition from the soil surface are 28%, 14%, and 58%, respectively, and the proportions of CH4 escaping via these pathways are 18%, 1%, and 81%, respectively.Figure SCO2,0. The fluxes are also sensitive to the ebullition rate constant, kE, and the constant, Kθ, in Equation SCO2,0, a large Ei following from a large Si in Equation kV, is unimportant at the high SCO2,0 value of our humose experimental soil; it will be more important at lower SCO2,0 values. The CO2 and CH4 fluxes are also sensitive to the ratio of CH4 to CO2 production, αCH4, and the root gas transmissivity, kT.Over the hundredfold range in values shown in Figure 44.1SCO2,0 and kV values are expected. The ricefield carbon economy—and hence SCO2,0—depends on the soil's initial organic matter content and on management of crop residues and organic manures  the presence of inorganic oxidants and (b) the stochiometry of methanogenic soil organic matter decomposition and the resulting proportions of CH4 produced from dispoportionation of acetate versus reduction of CO2 with H2  is in the form of HCO3−. Removal of CO2 from the soil close to root surfaces will tend to raise the soil pH , 82% of the dissolved CO Section . But the Section  gives va Geilfus  reviews 2 hydration–dehydration reactions, by which H2CO3 and hence HCO3− equilibrates with CO2 (HCO3− + H+ = H2CO3 = CO2 + H2O), are slow, and so may be rate limiting for the apolastic–symplastic pathway or degassing of CO2 into the aerenchyma or both. The presence of carbonic anhydrase (CA), which catalyses the reactions, in the apoplast is therefore an important question. Cytosolic CA is ubiquitous in plant tissues  and well below toxic levels. This indicates that the rate of CO2 venting through the roots would be sufficient to avoid toxic concentrations in root cells.Is the concentration of CO2 in the roots may have a growth stimulating effect in rice by facilitating anaplerotic production of organic acids for amino acid synthesis , which fixes CO2 into oxaloacetate and malate so providing carbon skeletons for the GS pathway. In principle, if other factors are nonlimiting, increased CO2 supply in the roots would allow greater N assimilation.In fact, the enhanced availability of CO2. An upper estimate of the size of this sink can be got from the rate of N uptake by the roots with the crude assumption that all the N is taken up as NH4+ and assimilated via GS and PEPC. From the plant growth rate 3 + 8H+) and so limit the impairment of cation uptake caused by acidification . Also, apparent 1st dissociation constant of H2CO3, K1 = 4.45 × 10‐7 mol dm‐3; saturating water pressure,PH2O = 5 kPa; gas constant, R = 8.314 dm3 kPa K‐1 mol‐1Fig. S1 Measured and modelled results for the second replicate with 4 plants per pot.Fig. S2 Measured and modelled results for the third replicate with 4 plants per pot.Fig. S3 Measured and modelled results for the first replicate with 1 plant per pot.Fig. S4 Measured and modelled results for the second replicate with 1 plant per pot.Fig. S5 Measured and modelled results for the third replicate with 1 plant per pot.Click here for additional data file.


Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by progressive cognitive and memory loss. The vicious circle between dysfunctional microglia and amyloid‐β (Aβ) is a crucial pathological event and accelerates the progression of AD. Herein, a zwitterionic poly(carboxybetaine) (PCB)‐based nanoparticle (MCPZFS NP) with normalizing the dysfunctional microglia and Aβ recruitment is established for the treatment of AD. Compared with the neural polyethylene glycol (PEG)‐based nanoparticles (MEPZFS NPs), the MCPZFS NPs significantly alleviate the priming of microglia by decreasing the level of proinflammatory mediators and promoting the secretion of BDNF. Most importantly, quite different from PEG, the PCB‐based NPs exhibit the behavior to recruit Aβ into microglia, which significantly enhances the Aβ phagocytosis. Moreover, the Aβ degradation is changed from the conventional lysosomal/autophagy to the proteasomal pathway in the presence of MCPZFS NPs. After the treatment with MCPZFS NPs, the Aβ burden, neuron damages, memory deficits, and neuroinflammation of APPswe/PS1dE9 mice are significantly attenuated in the brain. Therefore, the PCB‐based MCPZFS NPs have great potential to serve as an “Aβ cleaner” and provide a new insight into the therapeutic strategy for AD therapy. A microenvironment‐responsive zwitterionic nanoparticle (MCPZFS NP) is designed to normalize the dysfunctional microglia. The negative crosstalk between microglia and amyloid‐β (Aβ) was halted and the NPs exhibit the ability to recruit Aβ. The Aβ degradation is changed from lysosomal/autophagy to the proteasomal pathway. The NPs provide an applied horizon of zwitterionic materials and a new insight into the therapeutic strategy for Alzheimer's disease. For the treatment of AD, intense focus has been directed toward inhibiting the abnormal production and aggregation of amyloid‐β (Aβ). However, thus far, numerous failures in clinical trials indicate that targeting Aβ alone is far more enough.For the aspect of modulate the dysfunctional microglia, fingolimod has been proved to reduce the production of inflammatory mediators in microglia.p‐boronic acid benzyl)dimethylammonium bromide]  is chosen for siSTAT3 condensation and stimuli‐responsive release.d‐mannopyranoside (Man) is selected to guide the NPs to penetrate BBB and target to microglia.Nanoparticles (NPs) have the potential to overcome these shortcomings. Neutral materials such as polyethylene glycol (PEG)‐modified NPs have been commonly used to increase the stability and blood half‐time of NPs. However, modification of PEG reduces the efficiency of cellular uptake and endosomal/lysosomal escape of NPs and results in rapid clearance in vivo, which significantly restricts the therapeutic effects.FigureMan‐PCB‐PB/ZnO/fingolimod NPs, MCPZF NPs) via hydrophobic interaction to encapsulate fingolimod and ZnO in the hydrophobic region. The positively charged MCPZF NPs were condensed with siSTAT3 to form Man‐PCB‐PB/ZnO/fingolimod/siSTAT3 NPs (MCPZFS NPs). Then the performances of the MCPZFS NPs in cellular uptake, microglial regulation and AD mice therapy were investigated. Cellular and animal studies demonstrated that the MCPZFS NPs significantly normalized the function of microglia and relieved the AD symptoms compared with the PEG‐based NPs . Most excitingly, we discovered that the PCB‐based carrier could not only improve the cellular uptake and endosomal/lysosomal escape, but also recruit Aβ into microglia to enhance the Aβ clearance. Importantly, the MCPZFS NPs altered the Aβ degradation behavior through proteasomal pathway, which was quite different to that of MEPZFS NPs. Collectively, it was demonstrated that MCPZFS NP was also a promising “Aβ cleaner” for AD therapy, and provided a new horizon of zwitterionic materials for AD therapy.As shown in The therapeutic mechanism is illustrated in Figure 22.11H NMR spectra . For comparison, Man‐PEG‐PB polymers were also synthesized and confirmed . The hydrophobic ZnO NPs were synthesized via a previous methodThe synthesis route of the Man‐PCB‐PB copolymers is shown in Figure S1 (Supporting Information). The chemical structures of the monomer and polymers were characterized by The Man‐PCB‐PB and Man‐PEG‐PB polymer could self‐assemble via hydrophobic interaction to form NPs by encapsulating fingolimod and ZnO, which were abbreviated as MCPZF NPs and MEPZF NPs, respectively. The diameter of MCPZF NPs was 84.0 nm, and MEPZF NPs had a comparable diameter of 89.4 nm . The zeta potential values of them were 45.0 and 25.4 mV, respectively . This suggested that the ability of siRNA condensation for MCPZF NPs was superior to that of MEPZF NPs. To verify these, gel‐electrophoresis assay was adopted to evaluate the complex ability of siSTAT3. MCPZF NPs completely loaded the siSTAT3 at an N/P ratio of 7. However, MEPZF NPs could only completely bind siSTAT3 at higher N/P ratio of 11 . The results revealed that the complex capacity of MCPZF NPs was higher than that of MEPZF NPs. This might attribute to the cationic quaternary amine group of zwitterionic polymer PCB.FigureTo ensure a rational comparison of the two NPs, the N/P ratio of 11 was chosen for the following experiments. The diameters of the obtained NPs were about 100 nm, which were a little increased compared with that of the cationic NPs Figure2a. The−3m H2O2 solution for 1 h. The diameters of both MCPZFS NPs and MEPZFS NPs were significantly increased, which was also corroborated by TEM ‐labeled Aβ42 as a fluorescent probe. BV2 cells were incubated with Aβ42 in the presence of the NPs for 2 h and the MFI of cells were detected by flow cytometry. Co‐incubation with MCPZFS NPs resulted in a dramatic enhancement of Aβ phagocytosis compared with the Aβ group, whereas the treatment with MEPZFS NPs only slightly elevated the phagocytic uptake of Aβ Figure5a. NexThe intracellular distribution of Aβ and NPs in BV2 cells was further observed by CLSM. As shown in Figure Next, the Aβ degradation mechanism was examined to investigate the metabolic pathway of the NPs. In the presence of lysosomal/autophagy inhibitor bafilomycin A1 (Baf A1), the Aβ degradation of the free Aβ42 and MEPZFS group was almost completely inhibited, indicating that Aβ was degraded through the lysosomal/autophagy pathway with or without the treatment of MEPZFS NPs. However, Baf A1 could only slightly attenuate the Aβ degradation of MCPZFS group Figure d. The Aβ2.5The BBB permeability of MCPZFS NPs labeled with FITC in APPswe/PS1dE9 transgenic mice (APP/PS1 mice) was verified via a Kodak in vivo imaging system. In comparison with the nontargeted CPZFS NPs, MCPZFS NPs could enrich in the brain more effectively after 12 h injection . Moreover, the brain were sectioned and stained for microglia by using Iba1 antibody. As shown in Figure S14b (Supporting Information), the majority of MCPZFS NPs signal was localized with Iba1‐labeled microglia. The results indicated that the MCPZFS NPs could effectively deliver into brain and specially target to the microglia.FigureNext, encouraged by the results that MCPZFS NPs could enhance the Aβ phagocytic activity of microglia in vitro, we wondered whether Aβ phagocytosis were altered in APP/PS1 mice. After one week treatment, the amyloid plaques and microglia in the cortices were detected by using immunofluorescent staining. Compared with the other groups, a significant increase of Iba1‐labeled microglia (green) was observed adjacent to the 6E10‐labeled amyloid plaques (red) with the treatment of MCPZFS NPs  experiment was performed to assess the spatial learning and memory. During the 4 day training process, AD mice treated with MCPZFS NPs exhibited remarkable shorter escape latencies than that of PBS‐treated group, whereas the mice treated with free drugs, CPZFS NPs, and MEPZFS NPs only showed slightly improved Figure b. MoreovTo investigate the effect of the NPs on the Aβ burden of APP/PS1 mice, we detected Aβ deposits by using immuno‐histochemistry. The APP/PS1 mice treated with MCPZFS NPs significantly reduced the plaque area and numbers in the hippocampus and cortex compared with the PBS‐treated group and other groups Figure f. Moreov2.6FigureThe cytokines produced by microglia were crucial for the inflammatory process of AD, the levels of several important cytokines in the cerebral homogenates were measured by using ELISA kits. Several key proinflammatory cytokines including IL‐1β, Interferon‐γ (IFN‐γ), IL‐6, and interleukin 17A (IL‐17A) were elevated in the brain of APP/PS1 mice. As expected, the levels of the proinflammatory cytokines were notably reduced by the treatment of MCPZFS NPs compared with the other groups Figure7a,b. T3In this study, we present a microenvironment‐responsive zwitterionic nanoparticle (MCPZFS NP), which could recruit Aβ and normalize the priming of microglia by the synergistic effects of the carrier and drugs. The MCPZFS NPs could effectively permeate BBB and recruit extracellular Aβ to endocytose into microglia, which could act as an “Aβ cleaner” and cause a remarkably increase in Aβ phagocytosis. Moreover, it was discovered that MCPZFS NPs mediated Aβ degradation through proteasomal pathway due to rapid endosomal/lysosomal escape of PCB, which was quite different from the PEG‐based NPs (MEPZFS NPs) of lysosomal/autophagy pathway. Compared with the MEPZFS NPs, MCPZFS NPs significantly switch the dysfunctional microglia toward an alternative activation phenotype, which give rise to a reduction of the proinflammatory cytokines and ROS. After four weeks treatment of MCPZFS NPs, the Aβ burden, spatial learning and memory deficit, and neurologic changes of APP/PS1 mice were largely rescued. In conclusion, the study demonstrated that the MCPZFS NPs normalized the dysfunctional microglia via two synergistic approaches: 1) the fingolimod‐ and siSTAT3‐based inflammatory modulation and 2) PCB‐based Aβ recruitment of microglia. The synergistic strategy significantly hindered the crosstalk between microglia and Aβ, which attenuated the AD pathology. Therefore, the PCB‐based MCPZFS NPs provided a new horizon of zwitterionic materials for AD therapy as well as other neurodegenerative disorders.4Preparation and Characterization of the NPs: To prepare MCPZF NPs, a 100 µL solution of Man‐PCB‐PB, fingolimod and ZnO in methanol was added dropwise into 5% glucose aqueous solution (1 mL) under strong stirring. Then the methanol was removed by dialyzing against 5% glucose aqueous solution. The MEPZF NPs were prepared with the similar method as MCPZF NPs. Next, MCPZFS NPs and MEPZFS NPs were prepared by adding STAT3 small interfering RNA (siSTAT3) to the NPs solution at various N/P ratios at room temperature for 30 min, and the siRNA binding ability of NPs was evaluated by agarose gel retardation. The diameter and zeta potential of NPs were evaluated by dynamic light scattering instrument . The morphology of the NPs was measured by TEM (JEM‐2100 electron microscope). The drug loading of fingolimod for NPs was detected by high performance liquid chromatography (HPLC).In Vitro Release of Fingolimod: 2 mL of MEPZFS NPs and MCPZFS NPs in dialysis bag (MWCO 3500) was incubated in 50 mL of 0.5% Tween‐80 with or without H2O2 (1 × 10−3m) at 37 °C under horizontal shaking (150 rpm). At predetermined time intervals, 100 µL of the medium was removed and the same volume of fresh solution was supplemented. The concentration of fingolimod was detected using HPLC, which equipped with a UV detector and C18 column at 30 °C . For the HPLC analysis, the mobile phase was a mixture of 50 × 10−3m potassium dihydrogenphosphate (pH 3.0)/acetonitrile  at a flow rate of 1.0 mL min−1. The concentration of fingolimod was determined at the retention time of 2.7 min at 226 nm.Transportation across the In Vitro BBB and Cellular Uptake of NPs: To mimetic the BBB, bEnd.3 cells were seeded in the upper chambers (pore size: 0.4 µm) of a transwell model. After the resistance value reached 200 Ω cm2 of bEnd.3, BV2 cells were seeded on the lower chamber. Next, the bEnd.3 cells and BV2 cells in the transwell model were incubated with 10 × 10−6m Aβ42 oligomer for 48 h. NPs containing 1 µg Cy5‐siRNA were added into the upper inserts. After 2 h incubation, the mean fluorescence intensity of FAM in BV2 cells was assessed using BD Caliburflow cytometry .Endosomal/Lysosomal Escape of NPs: 2 × 105 BV2 cells were seeded in 35 mm Petri dishes (Cellvis) for 24 h, and then incubated with NPs including 1 µg FAM‐siRNA for 2 and 6 h at 37 °C, respectively. The cells were washed three times with PBS followed by staining with LysoTracker Red DND‐99 for 45 min at 37 °C. The cells were then washed three times with PBS and fixed with 4% paraformaldehyde for 15 min at 37 °C. Finally, the nucleus was stained with DAPI for 15 min at 37 °C. The fluorescence images were taken by CLSM .Aβ‐Induced Cytotoxicity Assay: Aβ‐induced cytotoxicity was determined by the MTT assay. BV2 cells were seeded in 96‐well plates with about 1 × 104 cells per well. Aβ42 (10 × 10−6m) with or without the various NPs was added to the cells and the concentration of fingolimod in NPs was 200 × 10−9m. After 24 h incubation, 20 µL MTT (5 mg mL−1) was added to the wells for another 2 h incubation. Subsequently, the medium was replaced with 100 µL DMSO each well. The absorbance was determined at 490 nm using a Tecan microplate reader .Downregulation of p‐STAT3: BV2 cells cultured in six‐well plates were treated with different NPs for 48 h, followed by Aβ42 (10 × 10−6m) incubation for 8 h. After that, the cells were washed by PBS and then lysed for total protein extract. The level of p‐STAT3 was detected by Western blot analysis and β‐actin was used as the loading control. The western blot analysis was performed at least twice.Aβ‐Induced ROS and Inflammatory Cytokine Detection: Primary microglia cells cultured in 35 mm Petri dishes (Cellvis) were treated with 10 × 10−6m Aβ42 in the presence or the absence of NPs for 12 h. The concentration of fingolimod in NPs was 200 × 10−9m. After that, the cells were labeled with 10 × 10−6m DCFH‐DA at 37 °C for 20 min. Then, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min at 37 °C. Subsequently, the nucleus was counterstained with DAPI at 37 °C for 15 min. Then, the fluorescence images were taken by CLSM . Alternatively, the cells were also collected to detect the fluorescence by BD Caliburflow cytometry . The supernatants after 12 h incubation were collected and levels of TNF‐α, IL‐1β, and BDNF were detected by using the ELISA kits according to the procedures provided by the manufacturers.Aβ Phagocytosis and Degradation Assay: To evaluate the effect of the NPs on Aβ phagocytosis, BV2 cells were incubated with FITC‐Aβ (500 × 10−9m) with or without NPs for 2 h. The fluorescence of BV2 cells were detected by flow cytometry analysis. For Aβ degradation, BV2 cells were washed three times after the 2 h incubation, and then cultured in Aβ‐free media with or without Baf A1 and MG132 for an additional 4 h. The fluorescence was analyzed by flow cytometry.g for 10 min. The fluorescence of the resuspended precipitates was quantified using microplate reader  with the excitation wavelength of 488 nm and emission wavelength of 525 nm.To further investigate the effect of NPs on Aβ phagocytosis and degradation, BV2 cells were treated with FITC‐Aβ and NPs containing Cy5‐siRNA for 1 h. Then, the cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min at 37 °C. The nucleus was stained with DAPI at 37 °C for 15 min and then the distribution of Aβ and NPs in BV2 cells were observed by CLSM . Furthermore, FITC‐Aβ42 and NPs were incubated at 37 °C for 5 min, followed by centrifuging at 14 000 × In Vivo Biodistribution: All animal experiments in this article were carried out in accordance with guidelines evaluated and approved by the ethics committee of Tsing Hua University, Beijing, China. To investigate the BBB permeability and microglia targeting ability, FITC‐labeled CPZFS and MCPZFS NPs were administrated intravenously to APP/PS1 mice. After 12 h of treatment, the mice were sacrificed and the organs  were excised. The biodistribution of mice were detected via a Kadak in vivo imaging system. After the biodistribution studies, the brains were sectioned and stained for microglia by using Iba1 antibody.Drug Treatment of APP/PS1 Mice: APP/PS1 mice (8 month old) were treated with NPs at the fingolimod dose of 0.5 mg kg−1 intravenously every 2 days. The mice of WT and AD control groups were treated with PBS. For studies of microglial phagocytosis of Aβ, the mice were injected for one week. For behavioral tests and therapeutic effect, mice were injected for four weeks.Morris Water Maze Experiments: After four weeks of treatment, the mice were tested in a MWM . A 10 cm platform was fixed to 1 cm beneath the surface of water, whereas the starting position was counterbalanced. During the first 4 days, the animals were tested four times daily from four different positions around the border of the maze and they were allowed to swim for 90 s to find the platform, on which they were allowed to stay for 5 s. If the animal unable to find the platform within 90 s, it was guided and kept on the platform for 5 s. On the fifth day, the mice were tested for memory retention in a probe trial with the platform removed. Each of the mice was allowed to swim 90 s from two starting points far away from the platform.Immunostaining and Nissl Staining of Brain Tissue: After the MWM test, mice were anesthetized, intracardiac perfused with PBS, and then sacrificed. The whole brains were harvested and fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 4 µm. 6E10 antibody for amyloid plaques was performed to detect the amyloid burden. To examine the phagocytosis of Aβ, 6E10 and Iba1 antibody was employed to determine the colocalization of amyloid plaques and microglia. For Nissl staining, the brain sections were stained with cresyl violet to detect the neuronal injury in the hippocampus of mice.Western Blot Analysis and ELISA: The brain tissues were dounce‐homogenized in RIPA buffer, followed by centrifuging at 14 000 × g for 30 min at 4 °C and the supernatant was collected. Levels of Aβ monomer, Aβ oligomer and BDNF were analyzed by Western blot. The levels of IL‐6, IL‐17A, IL‐1β, and IFN‐γ were detected by using the ELISA kits according to the procedures provided by the manufacturers.Statistical Analysis: All data were expressed as mean ± SD. The significance was analyzed by the unpaired Student's t‐test or one‐way or two‐way ANOVA followed by Bonferroni post hoc test by using GraphPad Prism version 6.0 software.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


The increase in steric bulk caused by the knotting locks a crown ether onto the thread. Removal of the lutetium ion unties the knot, and when the axle binding site for the ring is deactivated, the macrocycle spontaneously dethreads. When the binding interaction is switched on again, the crown ether rethreads over the 10 nm length of the untangled strand. The overhand knot can be retied, relocking the threaded structure, by once again adding lutetium ions.We report on a rotaxane‐like architecture secured by the in situ tying of an overhand knot in the tris region of the axle through complexation with a lanthanide ion (Lu Macroscopic knots persist and have distinctive properties owing to intra‐strand mechanics and inertia . In contrast, at the nanoscale, every part of a knotted strand undergoes random thermal motion in all directions. Accordingly, extrapolating knot properties between such disparate length scales, as with machine mechanisms,1H+, Scheme L2 was treated with Lu(CF3SO3)3 in MeCN at 80 °C to generate overhand knot Λ‐L2⋅Lu .1H NMR spectroscopy ,1H NMR spectroscopy  confirmed that both the knotted conformation of the strand and the threaded structure are maintained in 1H+ , which is due to π‐stacking in the knotted conformation,f1 and Hf2 appear at δ=4.55 ppm in 1H+ .1H+ was “switched off” by deprotonation with triethylamine, forming 1 .2Cl2, the 1H NMR spectrum of 1 is broad for internal regions of the axle . X‐ray crystal structures of knotted tris lanthanide complexes1 is still kinetically stable; at room temperature, 1 showed no signs of dethreading over several weeks in CD2Cl2 or MeCN‐3d solution.To confirm that the overhand knot blocks dethreading of the macrocycle, the dibenzylammonium binding site of 1 was untied, and the macrocycle released, by treatment of the deprotonated rotaxane with tetraethylammonium fluoride (10 equiv) in CD2Cl2 , with no evidence of a demetalated pseudorotaxane. This indicates that with the binding site deactivated, ring dethreading occurs as soon as the knot is untied. The quantitative dethreading further confirms the complete deprotonation of 1 (the DB24C8–secondary ammonium equilibrium in CD2Cl2 strongly favours the pseudorotaxane).The overhand knot of L1H+⋅Lu with DB24C8 failed to generate any [2]rotaxane (Scheme S9).The presence of the knot also prevents threading onto the axle: Treatment of the protonated knotted thread Λ‐L1 in CD2Cl2 afforded the pseudorotaxane complex L1H+ over 48 h 3 (1.1 equiv in MeCN‐d3) was added, and the solution heated at 80 °C. After 16 h, both 1H NMR and ESI‐MS analysis confirmed the formation of 1H+ in approximately 90 % yield  and untying (<5 min at room temperature) the stopper knot is particularly striking. Such processes may prove useful in the development of functional knotted molecules and materials.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The electro-depositions were completed at 20 min at a stirring rate of 150 rpm at temperature of 50 °C and pH of 4.The effect of S. tuberosum (ST) as bath additive at varied interval of 5–25 ml to the coating properties was noted. Electrodeposition parameters were constant at a voltage of 3.5 V for Zn-based coatings. The outline of bath condition as it influences the microhardness and wear rate were set into consideration. Hence, the coating microhardness and wear rate at constant electrodeposition parameters and varied ST were acquired. Hence, liquid fluid additives can be used for performance of fabricated coatings in advanced surface engineering application.In this study, the effect of the incorporation of composite and eco-friendly particles to develop new engineering materials on the developed zinc electrolyte containing TiO Specifications tableValue of the data•The given data will give demonstration to surface engineering specialists on the connection and effect of ST on zinc based electrolyte and the continuous metal matrix induced electrolyte in a given engineering components.•The data could be used for investigating the effect of eco-friendly biodegradable solanum tuberosum juice on the coating microhardness and wear rate progression for advanced engineering application.•The data obtained can be used in investigating the strengthening behaviour of ST in a zinc based electrolyte relating to its mechanical characteristics.12/TiB2/ST on wear and microhardness data was collected.The microhardness and wear rate of the fabricated coatings at constant process parameters were collected and the experimental outline of the work data was generated and represented via plots. Electrodeposition took place at 50 °C temperature for 20 min at a stirring speed of 150 rpm. 2, Zn–TiB2 and Zn–TiO2–TiB2 with variation addition of Solanum (5 and 10 L) at constant current density. From the collected data, it was observed that the addition of ST on Zn–TiB2 and Zn–TiO2 and Zn–TiO2–TiB2 composited shows improved microhardness properties. Maximum hardness value of 199.6 HV0.1 was obtained at Zn–TiO2–TiB2–10ST. These highest microhardness properties hence can be linked to the network of natural eco-friendly additives participating at the formation of the coatings 2, Zn–TiB2 and Zn–TiO2–TiB2 results were collected with much focus on the addition of Solanum additive on the composites coatings. Solanum tuberosum gives better enhancement in anti-wear properties which is supported by result observed from 2S. tuberosum tuber of equivalent weight of 15 g were selected, peeled, washed, and sectioned into smaller pieces. Then the smaller pieces were squeezed into deionized water to remove the fluid. The mined juice was stored in clean bottles and refrigerated. The S. tuberosum tuber used was shopped from Pretoria, South Africa A plane low carbon steel sheet of 60 mm × 60 mm dimension, with the thickness of 1 mm, was used as a substrate in this research. Other used materials include a zinc plate anode (99.9% pure) and grinding paper in the order of 60 μm, 120 μm, 400 μm, 800 μm, and 1,600 μm for surface preparation. An electrodeposition bath solution was prepared using distilled water. Samples were activated by sinking them into a 2 M HCl solution for 10 s, and then rinsing them in distilled water, which is in accordance with Popoola et al.


Saccharomyces cerevisiae G1-specific axial budding gene AXL2 is detected throughout the cell cycle and its phase-specific expression is lost. Promoter replacement abolished cell cycle-dependent RNA degradation and rendered the RNA insensitive to the deletion of nuclear ribonucleases. Together the data reveal a model of gene regulation whereby RNA abundance is controlled by promoter-dependent induction of RNA degradation.Cell cycle progression depends on phase-specific gene expression. Here we show that the nuclear RNA degradation machinery plays a lead role in promoting cell cycle-dependent gene expression by triggering promoter-dependent co-transcriptional RNA degradation. Single molecule quantification of RNA abundance in different phases of the cell cycle indicates that relative curtailment of gene expression in certain phases is attained even when transcription is not completely inhibited. When nuclear ribonucleases are deleted, transcription of the  Catala and Abou Elela show that the nuclear RNA degradation is executed in a promoter-specific manner, ensuring cell cycle-dependent gene expression. This study illustrates that local gene expression can be temporarily regulated without affecting overall transcription. This cell cycle requires the synthesis of a panel of proteins required for replication, many of which are expressed only when they are needed. Consequently, progression through the cell cycle is accompanied by dramatic reorganization of gene expression that is often referred to as cell cycle-regulated transcription3. 20% of yeast genes are expressed in a cell cycle-dependent manner, guided by the activation of transcription factors and cycling genes4. These waves of expression are often divided into four events that take place in G1, S, G2/M, and in the transition between the M and G1 phases. Genes associated with these waves are classified into clusters based on expression patterns and promoter motifs that promote cell cycle-regulated transcription3. Furthermore, shared common promoter elements are co-regulated by specific subsets of transcription factors, like the G1/S SCB-binding factor and MCB-binding factor6. These promoters can be used experimentally to induce phase-specific expression of reporter proteins8.Cells proliferate through a well-coordinated cycle ensuring the replication and redistribution of all cellular components9. Metabolic RNA labeling and dynamic transcriptome analysis identified hundreds of genes in budding yeast with periodic changes in, transcription and mRNA degradation, rates13. In general RNA degradation follows peaks of mRNA synthesis at defined times during the cell cycle. Strikingly, it was shown that the promoters of two mitotic genes (SWI5 and CLB2) trigger cell cycle-dependent RNA degradation in the cytoplasm14. In these two cases, it was proposed that RNA is marked for decay by factors loaded on the RNA during transcription, leading to cytoplasmic degradation at the end of the cell cycle. However, it is not clear how the factors are recruited to the promoter, nor how the RNA is degraded in the cytoplasm. It is also unclear if co-transcriptional RNA degradation in the nucleus contributes to cell cycle repression. A few examples of targeted nuclear RNA degradation have been linked to nutritional changes and other stresses17. However, it remains unclear generally how nuclear RNA degradation contributes to transcriptional regulation or how it regulates specific cell cycle genes.More recently, cell cycle changes have been associated with changes in RNA stabilityAXL2 during the cell cycle using single molecule analysis to determine the location, timing, and mechanism of phase-specific gene expression. Like many cell cycle genes AXL2 is thought to be expressed in the G1 phase of the cell cycle in a promoter-dependent manner19. However, in vitro studies suggested that this gene might also be regulated post-transcriptionally20. Here, we demonstrate that despite the sharp decline of AXL2 transcripts at the end of G1, it is transcribed albeit at a lower level throughout the cell cycle; live cell analysis and chromatin immunoprecipitation of RNA polymerase II confirmed that transcription of AXL2 continues in the S and G2/M phases of the cell cycle. Strikingly, deletion of nuclear ribonucleases completely blocked the characteristic periodicity of mature AXL2 mRNA levels. Ribonucleases were associated with the chromatin linking the RNA degradation machinery to transcription. Consistently, substitution of the AXL2 promoter impaired the cycling of gene expression. Together our data reveal a model of gene expression, in which cell cycle expression is achieved through promoter-dependent RNA degradation.Most biochemical assays provide estimates of average RNA amounts in a population of cells but cannot distinguish between nuclear and cytoplasmic RNA degradation. In such assays it is difficult to determine the location and timing of gene expression and the resolution of the expression cycle is often blurred by stochastic differences between cells. In this study, we monitored the synthesis and degradation of the phase-specific axial budding gene AXL2 using single-molecule fluorescence in situ hybridization (smFISH)21. AXL2 expression is required in the G1 phase of the cell cycle, and defects in its expression are easily scored by changes in budding patterns18. The Axl2 mRNA was detected using two independent sets of single labeled 20 oligodeoxynucleotides probes  and completed RNAs (hybridizing to both 5′ and 3′ end probes in the cytoplasm) per cell. Both nascent and full-length RNA decreased as the cells exited from the G1 phase of the cell cycle  at the AXL2 locus to accumulation of its RNA.Examining the expression pattern of Axl2 during the cell cycle using either the 5′ or the 3′ end probe reproduced the established expression pattern of Axl2 mRNA, which peaks in G1 and is repressed in later phases Fig. 18. We necle Fig. . In genecle Fig. . Notablycle Fig. . To veriAXL2, did not decrease but instead, increased in the S and G2/M phases. On the other hand, the association of RNAPII with the AXL2 open reading-frame was modestly reduced but continued to be detected in the S and G2/M phases. This data clearly indicate that Axl2 mRNA transcription continues beyond G1. Consistently, monitoring the accumulation of Axl2 mRNA in the same synchronized culture by RT-qPCR once again indicated that Axl2 mRNA abundance decreases but remains detectable in S and G2/M  and one endoribonuclease (Rnt1p). Rrp6p is a component of the nuclear exosome implicated in the 3′–5′ end degradation of non-polyadenylated RNA with free 3′ OH24. On the other hand, Rat1p is a 5′–3′ exoribonuclease that degrades uncapped RNA with 5′ monophosphate involved in transcription termination25. Unlike Rrp6p and Rat1p that are exclusively nuclear, Xrn1p is a mostly cytoplasmic paralog of Rat1p, but it has also been linked to nuclear RNA degradation26. Finally, Rnt1p is a nuclear endoribonuclease that exits from the nucleolus to the nucleoplasm at the end of the G1 phase of the cell cycle and cleaves RNA with conserved NGNN stem loop structures including one in Axl2 mRNA20. Mutating each of the 4 ribonucleases individually increased the abundance of both nascent and complete RNA in S phases when compared to that detected in G1 or when compared to the wild type RNA in the same phase  increased the ratio of the RNA in G2/M phase compared to G1  increased the effect of the endoribonuclease RNT1 in G2/M but not in S phase  or cells expressing a mutation in the Axl2 mRNA stem-loop recognized by Rnt1p  and the catalytic activity of Rnt1p (rnt1∆ c)  Fig. . This clRNT1 in the strains carrying the stem-loop and exoribonuclease mutations (rnt1∆ rrp6∆ xrn1∆ LoopM) did not increase the abundance of nascent or full-length RNA in S phase  that blocks Axl2 mRNA cleavage in vitro Fig.  also incRNT1 deletion, we examined the impact of short-term inactivation of Rnt1p activity on the abundance of Axl2 mRNA and compared it to the effect of RNT1 deletion by qRT-PCR. As expected, the deletion of RNT1 (rnt1∆) had the most impact on Axl2, increasing its mRNA 4-fold 28, consistent with direct cleavage by Rnt1p . As indicated in Fig. rnt1-D245R). Indeed, impairing the catalytic activity of Rnt1p increased by 3 folds the enzyme association with AXL2 chromatin  inhibited Rnt1p association with AXL2 chromatin near the cleavage site, while increasing the interaction with promoter region  that were identified using high-throughput assay as targets for nuclear RNA degradation20. However, it remains to be seen if these degradation events act as the primary mechanism of gene regulation or function as fail-safe mechanisms to ensure clean transcription repression. Distinguishing between transcriptional repression and cotranscriptional RNA degradation of transcribed genes is challenging since both mechanisms reduce the amount of RNA and, as we show also, may both depend on the promoter sequence. In addition, bulk measurements of total RNA decay cannot easily differentiate between these two nuclear mechanisms since most mRNA is cytoplasmic. Live cell analysis coupled with single molecule counting and RNAPII ChIP assay, as we performed here, will be required to clearly establish the regulatory mechanism of these candidate genes. Comparing both untagged RNA FISH assay and live cells assay of tagged RNA increases confidence in the data obtained and reduces the chance of technique specific artefacts. In addition, comparing between conditions or ribonuclease deletion using the same probe set or tag reduces the chance of errors of these techniques. In any case, the findings in this study caution against the automatic interpretation of the dependency of gene expression on promoter sequence as evidence for transcription regulation.It is unlikely that this direct feedback between synthesis and degradation is restricted to 41. Indeed, deletion of exoribonucleases including Rrp6p increases RNA abundance in a Rnt1p cleavage site-dependent manner  at 30 °C. G1 arrest was performed by adding 5 µg/ml α-Factor to the culture and monitoring the arrest by looking at the budding index of sonicated culture samples. After 3 h of treatment, >95% were un-budded and cells were collected. S phase arrest was performed by the addition of 200 mM hydroxyurea powder to the cultures and monitoring the arrest as for G1. After 3 h of treatment, >90% were budded and cells were collected.All yeast strains were grown in yeast complete (YC) media lacking the appropriate nucleotide or amino acid when needed for plasmid maintenance  with 2 g/L dextrose. Cultures were grown at 26 °C to accommodate thermosensitive strains such as 4. Samples were collected 0, 5, 10, 20, 40 min after the addition of thiolutin.The G2/M arrest was performed by the addition of 15 µg/ml nocodazole from 1.5 mg/ml stock in DMSO to the cultures and monitoring the arrest as above. After 3 h of treatment, >90% of the cells were arrested with large buds and the cells were collected. Transcription arrests were performed in cells grown in YC media at 26 °C. The cultures were shifted to 37 °C for 20 min prior to the addition of 150 µM bathocuprioinedisulphonic acid followed 10 min later by the addition of 10 µg/ml thiolutin with 150 nM CuSO21. 50 ml cultures were grown to an OD600 of 0.4–0.6 and fixed for 45 min at room temperature by adding 32% electron microscopy-grade formaldehyde to a final concentration of 4%. Fixed cells were washed with buffer B , digested for 10–20 min in buffer B with 20 mM vanadyl ribonucleoside complex, 20 mM beta-mercaptoethanol and 125–375 U of lyticase adjusted for each strain to achieve >90% cell digestion in that time frame. Digested cells were washed with buffer B and stored at −20 °C in 70% EtOH. 1.5e7 cells were rehydrated in 2× SSC and prehybridized in 2× SSC 10% formamide for 1 h at 37 °C. Hybridizations were carried out in tubes overnight at 37 °C in a final volume of 100 µl containing 2× SSC, 10% formamide, 10% dextran sulfate, and 10 ng of each set of probes. Probe sets consisted either of 12 × 20 nucleotide-long DNA oligos complementary to the 5′ sequence of Axl2 mRNA labeled with DyLight 650, 12 × 20 nucleotide-long DNA oligos complementary to the 5′ sequence of Axl2 mRNA labeled with DyLight 550, or 4 × 20 nucleotide-long DNA oligos complementary to the internal spacer “2” sequence of the pre-ribosomal RNA labeled with Alexa 488 used as a hybridization control. Probes sequences were chosen using the Stellaris Probe Designer web tool and synthesized by Biosearch technologies. After hybridization, cells were washed once with 2× SSC 10% formamide, once with 2× SSC 0.1% Triton, and once with PBS. 2E5 stained cells were deposited on poly-lysine coated coverslips, allowed to settle for 30 min, and washed with EtOH prior to mounting on a slide with ProLong Gold with DAPI. Slides were sealed after an overnight curing at room temperature. Visualization was performed on a Zeiss microscope with an oil immersion alpha Plan-Apochromat 100×/1.46 objective. A Zeiss mRM camera was used and z-stacks of 20 images spaced by 200 nm were collected with Zen 2012 software. DAPI was illuminated for 200 ms with a 365 nm LED and viewed through a filter cube 62HE, DyLight 550 was illuminated for 2 s with an HXP lamp and viewed through a filter cube 43HE. DyLight 650 was illuminated for 4 s with a 625 nm LED and viewed through filter cube 77HE. For display purposes, the image stacks were deconvoluted using the Zen 2012 constrained iterative algorithm and the maximum projection was presented in the pictures.The yeast smFISH protocol was performed essential as previously described600 of 0.4–0.6, concentrated by centrifugation to an OD600 of 2 and spotted on 1.2% agarose pads made with growth media. Cells expressing MS2-AXL2 or AXL2-PP7 and with a plasmid containing MCP-GFP and PCP-td-tomato were grown and prepared likewise. The coverslips were sealed with petroleum. Slides were visualized in a 26 °C incubation chamber on a Zeiss spinning disk microscope with an oil immersion alpha Plan-Apochromat 100×/1.46 objective. An Evolve 512 camera with EM gain set at 500 was used and z-stacks of 5–7 images spaced by 500 nm were collected using the Zen 2012 software. GFP was illuminated for 100 ms with a 100 mW 488 nm laser set at 10–20% through a 500–550 nm emission filter. Td-tomato was illuminated for 100 ms with a 40 mW 561 nm laser set at 20% through a 598–660 nm emission filter. The stacks of images were converted to 2D images by maximum projection and exported with the Zen 2012 software. RNA molecules were counted manually using the profile analysis tool in Zen 2012 on maximum projected images and scored when spots were twice as bright as the cellular background. Brightness and contrast were adjusted using Photoshop CS6 to set a common background level to the time series, for display purposes. The arbitrary cut-off was determined by contrast adjustment of live cell images to identify spots comparable in size to those observed by FISH. To compensate for the higher background of the live cell images in the GFP channel compared to the DyLight 550 and DyLight 650 channels of FISH we only counted GFP spots with SNR >2.Yeast cultures expressing MS2 repeats in the 5′ UTR of Axl2 mRNA and containing a plasmid encoding MCP-GFP and NLS-td-tomato were grown to an ODz-stacks of 20 images spaced by 200 nm were collected with Zen 2012 software. Calcofluor was illuminated for 100 ms with a 365 nm LED and viewed through filter cube 62HE. The images were deconvoluted with the Zen 2012 constrained iterative algorithm and the maximum projection was presented in the figures.2E6 cells fixed as for FISH were washed once with water and resuspended in 1 mg/ml Calcofluor and stained for 5 min at room temperature. Stained cells were washed twice with water and 4E5 cells were deposited on ConA coated coverslips, allowed to settle for 30 min, rinsed with water and mounted wet. Visualization was performed under a Zeiss microscope with an alpha Plan-Apochromat 100×/1.46 oil-immersion objective. A Zeiss mRM camera was used and 600 of 0.4–0.6 and 50 ml of culture was collected by centrifugation, washed with ice-cold water and resuspended in 300 µl LETS buffer  with 350 µl phenol and 750 µl acid-washed glass beads. Cells were broken by 7 cycles of vortexing for 30 s followed by 30 s on ice. The lysates were extracted 2 times with phenol: chloroform: isoamyl alcohol (25:24:1) and once with chloroform: isoamyl alcohol (24:1) before ethanol precipitation.Yeast cultures were grown to an ODrnt1⊗ cells in a total volume of 100 µl for 20 min at 30 °C. The RNA and 6 pmol of purified enzyme were premixed in reaction buffer  and the reactions were started by adding MgCl2 to a final concentration of 10 mM. Reactions were stopped by the addition of 1 volume of LETS buffer (described in the “RNA isolation” section) followed by phenol: chloroform: isoamyl alcohol (25:24:1) extraction.Cleavage reactions were performed on 25 µg of total RNA extracted from 32P randomly labeled DNA probes that were hybridized to the membranes overnight at 55 °C in 600 mM NaCl, 120 mM Tris, 8 mM EDTA, 0.2% Ficoll 400, 0.2% polyvinylpyrrolidone, 0.2% BSA, 0.1% SDS, 0.25 mg/ml fragmented herring sperm DNA, pH 8. Membranes were washed twice at 55 °C twice in 600 mM NaCl, 120 mM Tris, 8 mM EDTA, 0.1% SDS, pH 8 and once with 450 mM NaCl, 90 mM Tris, 6 mM EDTA, 0.1% SDS, pH 8, then exposed for 48 h to phosphoimaging screens and revealed on a GE Storm 860 scanner.RNA samples of 20 µg were separated on 1% agarose gels containing 120 mM formaldehyde in MOPS buffer and transferred by capillarity to a charged nylon membrane. Transferred RNAs were bound to the membrane by UV-crosslinking and revealed by methylene blue staining. PCR fragments were used as templates to synthesize 2 Mastercycler epgradient S.Contaminant genomic DNA was removed by DNase treatment of 25 µg of total RNA using 33 units of RNase-free DNase for 25 min at 37 °C on RNeasy spin columns. The treated RNA was eluted from the columns and 50 ng was used as template for reverse transcription using RT Transcriptor from Roche and random hexamers. The cDNA was diluted 15-fold in water and 3 µl of the dilutions were used as template for the qPCR reactions. The qPCR reactions were performed in 10 µl with BioRad iTaq Universal SYBR Green Supermix and 200 nM of primers in 96-well plates on an Eppendorf realplex600 0.4–0.6 and fixed for 20 min at room temperature by the addition of 1% formaldehyde to the culture. The crosslinking reaction was stopped by the addition of 3.6 mM unbuffered Tris and 360 mM glycine. The cell pellets were washed in ice-cold TBS , split into two aliquots and kept frozen at −80 °C. Frozen pellets were resuspended in 500 µl lysis buffer  with proteases inhibitors  and 500 µl acid-washed glass beads, and lysed using a Bertin Precellys 24 with 6 rounds of 10 s of shaking at 6500 rpm, spaced by 60 s pauses on ice. The lysates were adjusted to 750 µl with lysis buffer and sonicated 12 times for 10 s at 20% power amplitude with a Branson digital sonifier spaced by 2 min pauses on ice. The sonicated lysates were cleared by centrifugation at 13,000g for 10 min at 4 °C and 500 µl of cleared lysates were mixed with magnetic beads coated with the appropriate primary antibody and incubated overnight at 4 °C with rotation. For RNAPII ChIP, 50 µl of Pan mouse IgG Dynabeads  were coupled to 3 µg of purified mouse anti-RNA Polymerase II RPB1 antibody . For Rnt1p ChIP, 30 µl of M-280 sheep anti-rabbit IgG Dynabeads  were coupled to 3 µl of pre-cleared anti-Rnt1p antibody. The next day, the beads were washed twice with lysis buffer, twice with high salt buffer , twice with wash buffer  and once with TE . The de-crosslinking and elution of DNA was performed by re-suspending the beads in 100 µl TE containing 1% SDS and incubating overnight at 65 °C. Input DNA was de-crosslinked at the same time by incubation of samples of the cleared lysate diluted in TE containing 1% SDS overnight at 65 °C. The DNA was purified by RNase A and proteinase K treatments followed by 2 phenol:chloroform:isoamyl alcohol (25:24:1) extractions, chloroform:isoamyl alcohol (24:1) extraction, and ethanol precipitation with glycogen. The purified DNA pellets were resuspended in 10 mM Tris pH 8 and further diluted in water at 1:200 for the input DNA, 1:20 for the RNAPII ChIP, 1:10 for the Rnt1p ChIP.100 ml cultures were grown to ODsmFISH analysis: smFISH experiments were performed on 3 independent cultures and a target of 50 cells per phase were scored coming from each culture. In the figures, all data points are presented in the form of  violin plots unless stated otherwise in the legends and Supplementary Data 2 or larger than 20 µm2 were removed, while cells with a nucleolar pre-rRNA control signal were kept. Spots were detected and quantified for the 5′ and 3′ probes, then their intensity  and localization data was exported in a table format. After analysis of the 2D images, cells were manually assigned to G1, S, or G2/M phases of the cell cycle according to the bud size and nucleus position. The value of fluorescence intensity corresponding to 1 RNA molecule was established for each strain by taking the median value of the cytoplasmic RNAs of for all its dataset. The RNA molecule number per spot was calculated by dividing the spot fluorescence intensity by the value corresponding to 1 RNA molecule and rounding to the closest integer. As the primary object detection was based on nuclei identification, many G2/M cells had to be reconstructed from two cells identified from dividing nuclei. Nascent RNA per cell numbers were calculated from the brightest 5′ probes spot in the nucleus when more than 1 nuclear spot was detected. Cytoplasmic RNAs were scored when a spot detected with the 5′ probe also scored positive for the 3′ probe signal.RT-qPCR and chromatin immunoprecipitation analysis: Relative mRNA quantification was achieved through normalization to the Act1 mRNA using the formula: RQ = 2(CTWT−CTmutant)Axl2/2(CTWT−CTmutant)Act1, where CTs are the cycle threshold values determined by the Mastercycler software. PCR reactions were performed in technical duplicates or triplicates for cDNA produced from 3 independent cultures for both the wild-type (WT) and mutant strains. Relative enrichment of RNAPII or Rnt1p at the AXL2 locus was achieved through normalization to a region of the chromosome V using formula: RE = 2(CTinput−CTChIP)Probe/2(CTinput−CTChIP)ChrV. PCR reactions were performed in technical duplicates or triplicates for ChIP performed at least 2 times in independent cultures.Statistics and reproducibility: Statistical analyses of mean values of measurements were conducted using GraphPad Prism version 7.03 for Windows using Student’s t test if so stated in the figure legends with Welch’s correction for FISH data comparison, where variance between strains was unequal. The number of cells analyzed per FISH experiment, means and fold changes to wild-type with p-values are reported in Supplementary Data Further information on research design is available in the Reporting SummarySupplementary InformationDescription of additional supplementary itemsSupplementary Data 1Supplementary Data 2Supplementary Data 3