The structure of one of the first permanently porous metal phosphonates, MIL‐91(Al) was re‐determined using high resolution synchrotron powder X‐ray diffraction data. The new model is in a lower symmetry space group, with no disordered ligands in the structure, whilst remaining otherwise consistent with the reported compound. New milder synthetic conditions were also developed. In the intensely investigated field of porous materials, metal organic frameworks (MOFs) constitute a class of materials with exceptional properties and a remarkable structural diversity.2 is of great current interest due to increasing awareness of global warming,N,N′‐piperazinebis(methylenephosphonic acid)  and either Al3+ or Ti4+ as the framework forming cation [MOHn(H2L)]·xH2O .2 at low pressure as well as high selectivity for CO2, and is also thought to exhibit some structural flexibility during the CO2 adsorption process.Storage and separation of COThe present study reports a redetermination of the structure of the MIL‐91(Al), which removes the disorder present in the reported structure and may allow a deeper understanding of the nature of this flexibility.3:H4L). Under these conditions, the addition of base was not necessary. As a final optimization, the most crystalline samples were obtained when a heating and cooling ramp, each of 12 h, was used at the start and end of the reaction.Initial syntheses of MIL‐91(Al) followed the reported synthetic procedure.a = 18.959(9), b = 6.915(3), c = 11.256(6) Å, β = 90.54(2)°, V = 1467.8(13) Å3; C2/m].Rwp = 24.8 %; Supporting Information S7). The data were therefore re‐indexed in a unit cell with triclinic symmetry and approximately half the volume of the reported structure (V = 737.705(4) Å3, Rwp = 10.1 %; Table θ  was obtained as a microcrystalline powder consisting of needle‐shaped crystals. To re‐determine the structure of the compound, synchrotron X‐ray powder diffraction data were collected at beamline I11 , using the MAC detector to give the highest possible angular resolution.P1 and to remove the disorder of the linker molecules. Cell parameters and symmetry of this hypothetical structure were revised with PLATONP1 and were found to be in excellent agreement with the indexed values. A Rietveld refinement was then performed, applying chemically sensible restraints to bond lengths. Water molecules were located within the channels of the network by Fourier difference mapping, with sites O103 and O104 being disordered. The final cycles of refinement gave a good final fit to the data  are very similar, although the structure is rotated with respect to the unit cell axes . The asymmetric unit consists of two Al3+ cations, one in the center of the cell and one in the center of the bc faces of the cell, and two symmetry independent half H2L2– ligands. Each Al3+ cation is octahedrally coordinated by four oxygen atoms from four different phosphonate groups and two hydroxide ions. Al–OH–Al chains are formed through the corner‐sharing OH groups, which lie parallel to the a direction  Å; N21···H···O13–P11: 2.597(7) Å, indicated in Figure ···O: 2.43(3)–2.877(8) Å) and also with the O atoms of the CPO3 groups (O···O: 2.846(8)–2.864(10) Å).Structurally both the To confirm the synthesized compound has the same properties as the literature MIL‐91(Al) and to rule out the possibility of impurities, thermogravimetric analysis and elemental analysis measurements were performed. Thermogravimetric measurements (Supporting Information S3) indicate the loss of three water molecules per formula unit , in agreement with both the reported composition and that derived from the model from the powder X‐ray diffraction data. As previously observed, after the loss of water molecules, MIL‐91(Al) shows a step in the TGA  ascribed to the dehydroxylation of the framework. Elemental analysis results are also consistent with the literature MIL‐91(Al) (Supporting Information S1), confirming the high purity of the sample. These results allow us to rule out an impurity as the origin for the peak “splitting” observed.2 sorption isotherms at 77 K were recorded .Vmic = 0.150 cm3·g–1; determined using PLATON) also agrees well with the experimentally determined value. Additionally H2O sorption measurements were performed at 298 K, with MIL‐91(Al) shown to be porous to water with an uptake of 20 wt.‐% at 100 kPa . No substantial changes were observed in the diffraction patterns, indicating little or no degradation occurs during the measurements.To evaluate the porosity of the samples prepared by our new synthetic method, Nd Figure . MIL‐91 was developed. N2 adsorption experiments confirmed the porosity of the compound and indicate that the MIL‐91(Al) synthesized by the new method has a capacity for N2 consistent with the literature compound.Through the use of high resolution synchrotron powder X‐ray diffraction data, the MIL‐91(Al) structure was successfully re‐determined in the triclinic space group Supporting Information (see footnote on the first page of this article): Characterisation methods, synthesis, thermogravimetric analysis, IR spectroscopy, crystallographic methodology, summary of crystallographic results, comparison of Le Bail Fits of reported C2/m and new P1 MIL‐91(Al) structures and N2 isotherm of MIL‐91(Al).https://doi.org/10.1002/zaac.201600358 or from the author.Supporting information for this article is available on the WWW under Supporting InformationClick here for additional data file.


The formation of NiI versus NiII has been rationalised through the study of three different pathways: (i) halide abstraction by [Ni(PMenPhn)(3−)3], via an open‐shell singlet transition state; (ii) SN2‐type oxidative addition to [Ni(PMenPhn)(3−)3], followed by phosphine dissociation; and (iii) oxidative addition to [Ni(PMenPhn)(3−)2]. For the overall reaction between [Ni(PMe3)4], PhCl, and PhI, a microkinetic model was used to show that our results are consistent with the experimentally observed ratios of NiI and NiII when the PEt3 complex is used. Importantly, [Ni(PMenPhn)(3−)2] complexes often have little, if any, role in oxidative addition reactions because they are relatively high in energy. The behaviour of [Ni(PR3)4] complexes in catalysis is therefore likely to differ considerably from those based on diphosphine ligands in which two coordinate Ni0 complexes are the key species undergoing oxidative addition.Density functional theory (DFT) calculations have been used to study the oxidative addition of aryl halides to complexes of the type [Ni(PMe I intermediates or products from catalytic reactions,I in catalysis. Further investigation is essential to fully understand when and why NiI arises, and what its role is in catalysis, because this will have an impact upon the development of nickel‐catalysed reactions.The development of catalytic methods that use abundant, sustainable, and less expensive elements is an area of recent and intense focus. Nickel is one such element that has recently received increased attention.0 using [Ni(PEt3)4], it was proposed that electron transfer from [Ni(PEt3)3] to the aryl halide forms a solvent‐caged radical ion pair;I from NiII. This mechanism differs from that established for [Pd(PPh3)4], which proceeds via [Pd(PPh3)2] and a concerted three‐centre transition state.0 complexes bearing bidentate phosphine ligands.I and NiII products both do indeed arise via [Ni(PEt3)3]; NiI is obtained via an open‐shell singlet transition state and NiII is formed from an SN2‐type oxidative addition event  unless otherwise stated. Please see the Computational Methods for further details, and the Supporting Information for details of benchmarking. [Ni(PMe3)4] was used as a model for [Ni(PEt3)4] to reduce the computational cost and to prevent issues arising from the conformational complexity of PEt3; PMe3 has sufficiently similar properties to PMe3 to be an appropriate model.As a basis for developing a model for understanding structure/activity relationships in nickel catalysis, the oxidative addition of aryl halides to a model Ni3)4] spontaneously dissociates PEt3 to form [Ni(PEt3)3] (Keq≈10−3 mol L−1), with further PEt3 dissociation to form [Ni(PEt3)2] being possible (Keq<10−6 mol L−1).−1 for the first and second phosphine dissociation events, respectively, which is consistent with experiment . A relaxed scan of the potential energy as the Ni−P distance was increased to 4.0 Å identified no transition states for these dissociative reactions, but only a gradual increase in energy .In solution, [Ni2] and [Ni(PMe3)3] can each undergo oxidative addition to PhX . Barriers for the oxidative addition to [Ni(PMe3)2] are lower in all three cases , but the dissociation of PMe3 from [Ni(PMe3)3] is sufficiently endergonic to place these transition states higher on the free energy surface than those proceeding via [Ni(PMe3)3]. The most energetically favourable oxidative addition pathway is therefore the addition of PhX to [Ni(PMe3)3] 3] to form η2‐complexes 1. These readily undergo SN2‐type oxidative addition with very similar barriers . The barrier is not so dependent on halide identity, but the overall barrier for the process results from the decrease in entropy due to coordination of the aryl halide to the Ni0 centre.It is known from studies of oxidative addition to PdTS‐1–2‐Cl is depicted in Figure 0 centre acts as the nucleophile, attacking the ipso‐carbon in an SN2‐like transition state, rather than a concerted three‐centre transition state. The overall barriers for this process, with respect to [Ni(PMe3)4] and aryl halide, are 14.9, 15.0, and 16.2 kcal mol−1 for PhCl, PhBr, and PhI, respectively. While this is not the order of reactivity that might be expected, the energy differences are very small. Finally, 2 evolves to trans square planar complex 3 by dissociating a phosphine ligand and capturing the halide.Transition state 3)3] and [NiI(PEt3)3] were both isolated by Kochi,3)3] with the NiI product. The reaction proceeds via [Ni(PMe3)3(XPh)] (4). For PhI and PhBr, the aryl halide ligates the nickel via an electron pair on the halide  while for PhCl an η2‐complex is formed in which the ortho/meta edge of the arene coordinates the nickel . is obtained from the oxidative addition of PhBr to [Ni(PEt)4]. Similarly, an outer‐sphere electron transfer process was investigated using a Marcus theory treatment, but the barriers involved are far too high to be a plausible explanation for the observed reactivity (details can be found in the Supporting Information).An electron transfer mechanism was proposed by Kochi, so this was investigated first. One possibility is that there is a spin change within complex 0 to NiI . The nickel has a partially open‐shell, allowing the transfer of an electron from the (full) d‐orbitals to the interacting σ*CX orbital, simultaneously breaking the C−X bond. Finally, 5 dissociates phenyl radical to yield the experimentally observed [NiX(PMe3)3] complex (6). TS‐4–5‐I is shown in Figure The alternative is an open‐shell singlet transition state for halogen abstraction, with concomitant oxidation of NiI Figure . Transit−1 and so this pathway will be preferred over the oxidative addition mechanism described above. For PhBr, the barrier is 16.3 kcal mol−1, which is close to the energy of TS‐1–2‐Br . For PhCl, the barrier is much higher than the alternative oxidative addition transition state . These results are consistent with experimental observations; PhI leads primarily to NiI products, PhBr yields mostly NiII but some NiI, and PhCl yields exclusively NiII products.G=1.5, 3.7, 10.6 kcal mol−1 for PhI, PhBr, PhCl, respectively) is almost entirely due to a corresponding enthalpy change as the C‐X bond is broken .For PhI, the barrier is 10.0 kcal molTS‐4–5‐I and TS‐4–5‐Cl. This can be explained by the significant differences in the C−X bond dissociation energies (BDE), which are 66.9, 82.7, and 97.3 kcal mol−1 for PhI, PhBr, and PhCl, respectively.TS‐1–2 versus TS‐4–5) provide a good qualitative explanation for the behaviour of the three halobenzenes, but the quantitative agreement is limited. The application of a Boltzmann distribution to the relative barriers in Figures I : NiII selectivity of 0:100, 10:90, and 100:0 for PhCl, PhBr, and PhI, respectively. The available experimental values are, respectively, 0:100, 19:81, and 80:20. For the particular case of PhI, the experimental value is reported in toluene solution, but repeating the calculations in toluene did not yield a significant difference (see the Supporting Information). Better quantitative agreement between calculation and experiment can be obtained by taking into account the fate of the phenyl radical that is generated as a by‐product of NiI formation 4] and PhI in toluene was simulated initially ), phenyl radical trapping by the NiI complex (Eq. (4)), and the reaction of phenyl radical with the solvent (Eq. (5)). The barriers for Eq. (1) and Eq. (2) are taken from the calculations using toluene as the solvent, those for Eq. (3) and Eq. (4) are taken from calculated diffusion barriers, and the barrier for Eq. (5) was computed to be 10.6 kcal mol−1. The application of this microkinetic model yields a predicted NiI:NiII ratio of 83:17, which is in significantly better agreement with the value of 80:20 determined experimentally. Applying the same methodology to the reaction of PhBr gave a ratio of 8:92 .Halogen abstraction is very sensitive to the aryl halide, with a difference of 11.4 kcal mol‐1 between −1 for THF, 10.6 kcal mol−1 for toluene, and 10.8 kcal mol−1 for n‐hexane. This correlates well with the observation that the NiI:NiII ratio increases in the order n‐hexane< toluene<THF.The explicit introduction of a solvent molecule in the microkinetic model has the additional advantage of rationalising selectivity. The barrier for hydrogen abstraction is a function of solvent, with values that can be determined computationally: 9.5 kcal mol3)4], it should be noted that this type of complex is rarely applied in modern catalysis. The reactivity with PhI of a series of analogous complexes, bearing PMe2Ph, PMePh2, and PPh3 ligands was therefore evaluated using the same methods.While this study reveals the mechanistic details underpinning the observed halogen and solvent effects disclosed in Kochi's seminal study of oxidative addition to [Ni(PEt3)4] with aryl halides have been investigated previously by Cassar, who reported the rates of the reactions and their selectivities, but little analysis of the reaction products.3)4] reacts with PhCl, PhBr, and PhI to form [Ni(Ph)X(PPh3)2] and [NiX(PPh3)3] in varying proportions.I decreases in the order PhI>PhBr>PhCl. Interestingly, experimental evidence and computational studies showed that trans‐[NiX(Ph)(PPh3)2] decomposes rapidly to [NiX(PPh3)3] in the absence of excess PPh3.The reactions of [Ni(PPh3)] is far too high in energy to play a role in the reaction . However, [Ni(PR3)3] is readily formed in each case, particularly for PPh3 . Three possible pathways were then considered for the reaction of these complexes with PhI: (i) halogen abstraction by [Ni(PR3)3], (ii) oxidative addition to [Ni(PR3)3], and (iii) oxidative addition to [Ni(PR3)2].The equilibria for phosphine dissociation differ somewhat across the four ligand systems, but it is clear that in all cases [Ni3] and phenyl radical  is considerably lower than that of the corresponding [Ni(PR3)2] complex . Oxidative addition via [Ni(PR3)2] cannot therefore be completive with this pathway. Instead, the free energy profiles for halide abstraction must be compared with those for oxidative addition via the [Ni(PR3)3] complex 3] are high in energy , and much higher in energy than the halogen abstraction transition state. With the exception of the PMe3 system, the oxidative addition of PhI to [Ni(PR3)3] cannot compete with the halogen abstraction pathway, and therefore this reaction will occur exclusively via the latter pathway for PMe2Ph, PMePh2, and PPh3 complexes.In each case the energy of this transition state 3] complex. The [Ni(PR3)2] complex plays little, if any, role in the oxidative addition reaction when PMe3 is involved. In the specific case of iodobenzene, this holds true not only for PMe3, but for more commonly employed ligands such as PPh3.This study provides new insight into the mechanisms of oxidative addition of aryl halides to nickel complexes bearing monodentate phosphine ligands. Importantly, the key pathways—SI from Ni0 had not previously been reported, yet it clearly accounts for the major product in the reactions of aryl iodides with [Ni(PR3)4] complexes. Similar transition states have been shown to play a role in nickel‐mediated C−H functionalisation reactions where substrates have relatively low bond dissociation energies.The open‐shell singlet transition state for the direct formation of NiN2‐type oxidative addition of the aryl halide (via TS‐1–2) is influenced only slightly by the identity of the halide, while the open‐shell singlet transition state for halogen abstraction presents a more variable barrier that depends on the energy of the σ*CX orbital. The selectivity for NiI versus NiII is determined by the competition between both transition states and also by the reactions of the aryl radical with NiI or the solvent. From a computational perspective, the importance of considering open‐shell broken‐symmetry transition states and microkinetic models is demonstrated. The coordination number of the Ni0 intermediate is crucial; in contrast, most reactions of bisphosphine‐Ni0 complexes will proceed via complexes with only two phosphorus moieties attached, and will show rather different reactivity. Recent studies have shown that NiI complexes with bidentate phosphine ligands most likely arise as a result of comproportionation, rather than via electron transfer or halide abstraction.The pathway proceeding via Sn‐hexane) was accounted for using the SMD implicit solvation model.https://doi.org/10.19061/iochem‐bd‐1‐55). Kinetic simulations were carried out using the COPASI software package.All DFT calculations were carried out using Gaussian 09 (Rev D.01).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.


No change in the solution ORP was observed at the cathodic half-cell when both half-cells contain 5 M H2SO4, and Co3+ was formed in the anodic half-cell. An electro-scrubbing approach based on the above principles was developed and tested on gaseous-pollutants, CH3CHO and CCl4, by Co3+ and Co1+, respectively, with 90 and 96% removal achieved, respectively.Electro-scrubbing is being developed as a futuristic technology for the removal of air-pollutants. To date, only one homogeneous mediator for the removal of air pollutants has been generated in each experiment using a divided electrolytic flow cell in an acidic medium. This paper reports the concurrent generation of two homogenous mediators, one at the anodic half-cell containing an acidic solution and the other at the cathodic half-cell containing a basic solution. The concept was inspired by the change in pH that occurs during water electrolysis in a divided cell. A 10 M KOH electrolyte medium assisted in the electrochemical generation of low valent 14% Co Over the last decade, electrochemically oxidized metal ions, such as Ce4+, Ag2+ and Co3+, have been used as homogeneous mediators for pollutant degradation because of their very positive oxidation potentials, 1.4 V, 1.9 V and 1.8 V, respectively5, to generate what is considered as a futuristic technology by the United Nations Environmental Protection Agency (UN-EPA)6. In practice, these electrogenerated free metal ions, particularly Ag2+, are stable and soluble only under acid pH conditions (<1)7. Ce4+ and Co3+ have been generated in both divided and undivided cells, but Ag2+ can only generated in a divided cell because of its deposition as Ag metal on the cathode8.The increasing concentration of atmospheric air-pollutants has prompted intensive research into the development of new environmentally friendly and affordable degradation methods. The generation of homogeneous mediators in aqueous media plays a key role in the mediated electrocatalytic oxidation (MEO) of air-pollutant degradation2 evolution potential (0.0 V) is well below the standard redox potential of the low oxidation states or metals of Ce0 (−2.33 V) and Co0 (−0.28 V)9, which restricts reduction of the simple metal ions to lower oxidation states under acidic conditions10, although this may be possible under alkaline conditions. Interestingly, no systematic work has been undertaken to achieve lower oxidation state generation of soluble metal ions using an electrolytic cell without encountering problems with the water splitting reactions, although this feature is available in vanadium redox flow batteries which operate as a divided electrolytic cell in H2SO4 or CH3SO3H with a standard redox potential of 1.00 V (for V5+/V4+) and −0.26 V (for V3+/V2+)11. In practice, if electrolytic cells are to generate homogeneous mediators, they need to operate within the water splitting window and also at a potential far from the equilibrium value of their electrode reactions because of the inherent interfacial charge transfer and polarization resistances across the electrode-electrolyte interface12. In the case of the commercially available divided cell, this overpotential factor is substantial due to the properties of membrane separators like Nafion®13.In principle, reduced metal complexes could be useful for air-pollutant degradation. However, the H16. The following equations are relevant in acidic and alkaline media18:H+/H2 for every unit increase in pH17. The solution pH change also can alter some electrode potentials, and recently, a pH dependent water oxidation mechanism associated with a MnO2-modified electrode was reported19.Water-electrolysis using a divided electrolytic cell creates a pH gradient in each half cellet al. suggested that the use of either highly acidic or highly alkaline solutions can be used to favor the generation of an electrocatalyst in an electrochemical cell because of the potential window gain for the oxygen evolution reaction (OER) or hydrogen evolution reaction (HER)20. Hydrogen evolution is more facile when the concentration of protons is high, and oxygen evolution is easier when the hydroxide concentration is high17. Thus, in principle by combining the potential widening achieved by a pH change in both sides of an electrolytic cell, the simultaneous generation of both high and low oxidation state metal complexes  as two mediators for gas pollutant removal is facilitated.Morale-Guio IISO4 (0.01 M) in 5 M H2SO4 in the anodic part of the cell and 0.01 M [CoII (CN)5]3− in 10 M KOH in the cathodic part to generate the Co3+ electrocatalyst in acid and Co1+ in base, respectively. The advantage of using [CoII(CN)5]3− in the cathode cell is the ability to obtain a Co1+ electrocatalyst as a cyanide complex through a one electron reduction process in 10 M KOH medium without reduction to metallic cobalt; its pK value is approximately 18–19 21. In initial electrolysis experiments the pH and half-cell potential variations were monitored during the electrolysis of water with 1 M NaCl as the electrolyte. Co3+ and Co1+ generation was tested using the precursor, CoIISO4, with acid in both half-cells and with acid in the anodic half-cell and base in the cathodic half-cell. The combination of CoIISO4 in acid as the anolyte and [CoII(CN)5]3− in base and acid as the catholyte was also assessed for the simultaneous generation of Co3+ and Co1+. The electrodes selected for these experiments were a platinum coated titanium anode and a silver cathode. To assess the ability of electrochemically generated Co3+ and Co1+ to each remove one air-pollutant, the possibility of simultaneous electro-scrubbing of two gas pollutants CH3CHO and CCl4 was examined in a continuous mode of operation coupled with monitoring of the time dependence of the concentrations of pollutants with an online Fourier transform infrared (FTIR) gas analyzer. The results highlight the importance of pH and broadening of the potential window to generate two mediators simultaneously.The hypothesis that both high and low oxidation state mediators could be generated in one electrolytic experiment was tested in this study using the precursors, Co22. The changes in pH and half-cell potential during water-electrolysis in the divided electrolytic cell confirms that in a practical electrolytic cell with dual mediators it is advantageous to generate one of them in acid conditions in the anodic half-cell and the other under alkaline conditions in the cathodic half-cell.First of all, 1 M NaCl was added as the electrolyte to both half cells and the changes in pH and half-cell potential that occurred during electrolysis of water were determined. Figure IISO4 was placed in 5 M H2SO4 in both half cells, as applies in Fig. 3+ (reaction 5) was confirmed by conventional potentiometric titrations, and an oxidation efficiency of 39% was achieved. However, there was no change in the ORP, (−110 ± 10 mV reached in 1 min), in the cathodic half-cell during the 5 h electrolysis time implying no cathodic reduction occurred to give Co1+ (potentiometric titrations were not possible), and that dominant H2 evolution occurs, as shown in reaction 6.When CoIISO4 should be placed in 5 M H2SO4 in the anodic half-cell and in 10 M KOH in the cathodic half cell. The electrolysis results  and decrease to a minimum value of −978 mV in cathodic part, with −1200 mV being the standard potential21), consistent with the formation of Co3+ and Co1+ at the relevant half-cells during electrolysis. Although the generation efficiency of Co3+ was 41% in 5 h, the generation efficiency of Co1+ initially reached only 11% then decreased to 8%. In highly basic media (>6 M) Co2+ becomes [CoII(OH)4]2− 23, which may be reduced electrochemically to low-valent [CoI(OH)4]3−.As inferred from analysis of the above water-electrolysis experimental data, for dual mediator generation, 0.01 M Colts Fig.  now exhiIISO4 and [CoII(CN)5]2− are present in the anodic and cathodic half cells containing 5 M H2SO4 and 10 M KOH as the electrolytes respectively. The initial shift in the ORP of the electrolyzed solution at each half-cell to 1.35 V and −1.05 V in 30 min highlights the formation of higher oxidation state Co3+ and lower oxidation state Co1+ in the relevant half-cells. This result confirms that the pH change and potential widening effect can allow the generation of two homogenous mediators concurrently using a single electrolytic cell. A [CoII(CN)5]3− to [CoI(CN)5]4− reduction process occurs under highly alkaline conditions at Hg electrode as shown in equations E0 = −1.2 V (SCE) applying to 4 M and higher KOH concentrations24. A similar electrochemical reduction process is expected to occur at a Ag metal electrode with high KOH concentrations,Figure II(CN)5]3− in 5 M H2SO4, no change in the cathodic ORP occurred, as expected if no low-valent Co1+ formation had occurred 5]3− reduction can occur in the electrolysis conditions used above, experiments using cyclic voltammetry (CV) was undertaken in both acidic and basic media at a macrodisk Ag electrode 5]3− does occur (a reduction peak at −830 mV) in alkaline media  whereas H2 evolution is favoured under acid conditions 5]3− (not relevant to this paper) at about 0.65 V also is evident in curve b of Fig. data shows that when Co1+ and Co3+ are both generated simultaneously at the cathode and anode half cells, respectively, that the Co1+ yield increased when the current density increased from 20 to 30 mA cm−2 then decreased when a higher current density of 50 mA cm−2 was employed due to enhanced formation of inactive [Co(CN)5H]3−  of Co3+ reached 41% at the anodic half-cell after 5 h operation. With the help of the cathodic half-cell operation, an additional 15% generation efficiency of Co1+ formation was achieved to give a total of 56%, with this new approach.The efficiency of the generation of two mediators concurrently is an important practical consideration in an electrolytic cell. As shown in Fig. 28, the stability of the membrane was tested under the present harsh conditions. No prominent changes were observed in SEM images on either side of the membrane surface after electrolysis  using electro-scrubbing. Although the two mediators can be produced concurrently, quantitative assessment of air-pollutant removal by electro-scrubbing had to be undertaken by a stepwise approach due to practical limitations with the availability of only one online FTIR gas analyzer.Using the simultaneous generation of the Co1+ in the cathodic compartment under the optimized current density was used to degrade CCl4 in a continuous process. Note that Chloro Volatile Organic Compunds (CVOCs) like CCl4 cannot be degraded at the anodic half-cell due to electrode fouling by polymerization29. When the concentration of Co1+ reached 14%, ≈8 ppm of CCl4 was injected at a gas flow rate of 0.2 L min−1 through the bottom of the scrubber at a liquid flow rate of 3 L min−1 with a closed loop catholyte circulation. As shown in Fig. curve a, a rapid decrease in the outlet CCl4 to 0.3 ppm was observed and this trend was maintained for up to 1 h of operation, which is equal to 96% removal or 0.53 mg h−1 of CCl4 removed and 12.78 mg day−1 can be achieved if the same removal rate continues. In the absence of Co1+ in the electrolyte, the direct electrocatalytic reduction (DER) of CCl4 does not occur in the electrolyzed 10 M KOH solution. In this case  an initial decrease is detected in the outlet followed by an increase with time, and ultimately returning to the level of the inlet (≈8 ppm), which equates to 0% removal. These data confirm that the CCl4 removal process follows an MER pathway, as shown in reaction 9.The electrolytically-formed Co4 removal by electro-scrubbing for I hour are chloride (Cl−) in solution and CO2 in the gas phase. In brief, the chloride concentration in the catholyte solution phase increases from about 1 to 1.66 g L−1 as shown in Fig. 2 concentration in the gas phase increases to around the 30 to 40 ppm level and all CCl4 is removed as shown by increase in the gas phases FTIR CO2 stretching response in the 2289.9 to 2385.5 cm−1 range and a decrease in the CCl4 FTIR stretching response in the region 737.12–815.74 cm−1 (reference made to standard FTIR gas phase spectra available in the MIDAC library).Additionally, the byproducts identified to accompany CCl3+ was used to degrade CH3CHO through a continuous process. When the Co3+ concentration reached 41% by electrolytic generation, ≈30 ppm of CH3CHO was injected at a gas flow rate of 0.2 L min−1 through the bottom of the scrubber at a liquid flow rate of 3 L min−1 with closed loop circulation. As shown in Fig. curve a, a rapid fall in the outlet CH3CHO to a concentration of 3 ppm  occurs with this pattern continuing for up to 1 h of operation. The possibility of direct electrocatalytic oxidation (DEO) of CH3CHO (without presence of Co3+) was considered in an electrolyzed solution of 5 M H2SO4 . In this case, the initial decrease in the outlet concentration of CH3CHO ceased after 35 min, due to electrode fouling during DEO of CH3CHO25. In contrast, continuous removal of CH3CHO was achieved in the presence of electrogenerated Co3+ which confirms the removal process follows the MEO pathway without any additional reactions as shown in equation In an anodic half-cell, electrolytically generated Co4) at the cathode and one (CH3CHO) at the anode was achieved simultaneously with efficiencies in both cases being greater than 90%.In this investigation, widening of the potential range available by controlling the conditions used for water electrolysis has been utilised to generate two mediators simultaneously that facilitate effective air-pollutant removal, with one formed in each half cell. In a practical demonstration of the dual mediator generation approach, the degradation of one pollutant (CClII(CN)5]3−) was synthesized according to a literature procedure30. Briefly, 53.8 g of potassium cyanide dissolved in 50 ml water was added to 60 ml of a cooled solution (4 °C) containing 40.0 g of CoII(NO3)2 under a nitrogen atmosphere , after which an equal volume of chilled alcohol was added. The resulting mixture was chilled slowly until thin violet platelets of the cobalt cyanide complex appeared. The solid was collected by rapid filtration, washed with cold alcohol, dried in a vacuum desiccator, and then stored in an air-tight brown bottle.The cobalt cyanide complex . The electrode assembly was clamped tightly with Ti end plates using a filter press technique. Separate channel paths were available in each compartment through which the anolyte and catholyte solutions could flow across their relevant electrodes. A 0.2 L solution of CoIISO4 (10 mM) in 5 M H2SO4 and 0.2 L of each of CoIISO4 in 5 M H2SO4, CoIISO4 in 10 NaOH, [CoII(CN)5]3− (10 mM) in 5 M H2SO4 or 10 M KOH were placed in separate anolyte and catholyte tanks, respectively. The anolyte and catholyte solutions were circulated continuously through the anode and cathode compartments of the electrochemical cell at a constant flow rate of 70 ml min−1 using peristaltic pumps . The active Co1+ and Co3+ mediators were generated galvanostatically by applying constant current densities between 10 to 50 mA cm−2 using a DC power supply (Korea Switching Instruments). The effective surface area of each electrode exposed to the solution was 4 cm2. All measurements were carried out at 20 ± 2 °C.The electrolytic flow cell used for the simultaneous generation of Co2 of Teflon tubes was attached to top of the anolyte and catholyte tanks respectively, which were already attached to the flow through electrolytic divided cell, as shown in Fig. 2 and an electrolyte volume of 500 ml. The scrubbing system consisted of an air supply system, a scrubbing solution ([CoII(CN)5]3−/[CoI(CN)5]4− in a KOH solution or Co2+/Co3+ in H2SO4 solution), a scrubbing reactor column, and an FTIR gas analyzer system  equipped with a data logger. The anolyte and catholyte solutions were circulated continuously at a flow rate of 2 L min−1 using magnetic pumps . For electro-scrubbing, the activated solutions were pumped separately into the scrubber column at a flow rate of 3 L min−1. CH3CHO and CCl4 gases (from RIGAS (1000 ppm), Korea) and air mixtures, which were obtained by the controlled mixing of air and CH3CHO or CCl4 gas using mass flow controllers (MFCs), were introduced to the relevant bottom of the scrubbers at a set gas flow rate. The CH3CHO or CCl4 gas to air ratios obtained using the MFCs were confirmed prior to undertaking the experiments. The scrubbing solution was re-circulated through the electrochemical cell to regenerate Co1+ or Co3+. Before starting the CH3CHO or CCl4 removal experiment, the electrochemical cell was operated until Co1+ or Co3+ conversions reached nearly a steady state, and the scrubbing solution was then pumped into the scrubber column.During gas-pollutant removal, a scrubber column (40 cm high and 5.5 cm (i.d.)) packed with 1 cmCyclic voltammetry was carried out with a PARC VersaSTAT 3 instrument in a glass cell furnished with two compartments separated by a membrane. A platinum mesh and a dip type Ag/AgCl electrode were used as the counter and reference electrodes, respectively. Ag was used as working electrode, and were placed along with reference electrode in one of the cell compartments (acid or base as appropriate), while the counter electrode was placed in the other compartment.1+ were drawn periodically during the course of electrolysis experiments and the generated Co1+ concentration was calculated by titration against a standard [FeIII(CN)6]3− (1 mM) solution. The redox potential of the Co1+-containing solution was measured using an oxidation reduction potential (ORP) electrode  with data obtained with a pH/ISE meter  used to identify the end point during the titration. During each titration, [FeIII(CN)6]3− addition was stopped when the measured ORP value, which shifted to negative values with increasing Co1+ concentration, reverted to its initial value measured before electrolysis. The concentration of Co1+ was calculated from that of [FeIII(CN)6]3− consumed. In a similar manner, the Co3+ concentration from the anode compartment was obtained by titration with FeIISO4 (1 mM). Here, the initial ORP value of the Co3+-containing solution, which was approximately 600 mV, became increasingly positive with increasing Co3+ concentration. The concentration of Co3+ was calculated from that of the FeIISO4 titrant. The concentrations determined in this manner were reproducible to ±0.02 mM.Aliquots (5 ml) of the catholyte containing CoThe procedure used is based on that described in the user’s manual provided by HACH with their Model DR-2800 spectrophotomer. 5 ml of sample drawn from the electrolysis solution (catholyte) was neutralized with perchloric acid by titration and the resultant solution centrifuged to remove any precipitate present. If required, a final adjustment of the pH to 6–7 was made using 1 M NaOH. 0.8 ml of mercury thiocyanate (HACH reagent) and 0.6 ml of 1 M ferric perchlorate were then added to give a brownish yellow solution. The chloride content of this solution was then measured colorimetrically and the finally reported value determined after correction for dilution.Supplementary Information


Since the experimental realization of graphene,3) was proposed by Li et al.3  and MPSe3  were explored by Du et al.3  were obtained and characterized in experiments. The previous reports indicated that the band gaps of these MPX3 bulks range from 1.3 to 3.5 eV,Recently, a new kind of 2D materials  and MPSe3  monolayers to screen more proper photocatalysts for water splitting. Then the carrier mobility was calculated through deformation potential (DP) theory for MnPSe3 monolayer which is a direct‐band‐gap semiconductor and has strong absorption in the visible‐light region. The high carrier mobility of MnPSe3 monolayer (up to 625.9 cm2 V−1 S−1) could be comparable to or even higher than those of many other 2D materials, indicating that the transfer of carriers to reactive sites would be easier in the photocatalytic process. Our results disclose that MnPSe3 monolayer would be a promising photocatalyst for water splitting under visible light.Hydrogen generation by photocatalytic water splitting would present a promising method for solar energy conversion and play a very important role in solving serious environmental problems.3  and MPSe3  monolayers were explored first. To determine the ground state of MPS3  and MPSe3 , both spin‐unpolarized and spin‐polarized computations were performed. The results show that spin‐polarized total energies are less favorable than spin‐unpolarized ones for FePS3, CdPS3, ZnPS3, and FePSe3 monolayers, indicating that these monolayers have nonmagnetic ground states. However, MnPS3, NiPS3, and MnPSe3 monolayers prefer antiferromagnetic (AFM) coupling, which is more stable than the ferromagnetic (FM) state. The structures of these kinds of monolayers are similar, as shown in FigureThe structural properties of MPS3  and MPSe3  monolayers, which reveals that these monolayers are dynamically stable configurations and could be realized in experiments.To check the stability of the monolayers, the phonon spectra along the high‐symmetry points in Brillouin zone were computed, and are shown in Figure S1 (Supporting Information). There are no imaginary phonon modes in the phonon spectra of MPSC2D = /S0, where Etotal, ε, and S0 represent the total energy per unit cell, uniaxial strain, and the area of the optimized unit cell, respectively. The calculated in‐plane stiffness is shown in Figure S2 and Table S1 of the Supporting Information. The in‐plane stiffness of sulfides is higher than that of the corresponding selenides, which might result from the greater electronegativity of sulfur. Through the elasticity theory, an estimate for the typical out‐of‐plane deformation h/l ≈ (ρgl/C2D)1/3,l is the length of the monolayer, ρ is the density, and g is the gravitational acceleration. Assuming the length of the monolayer l to be 100 μm, the ratio of the vertical deformation and the length h/l is shown in Table S1 of the Supporting Information, which indicates that MPS3  and MPSe3  monolayers have sufficient rigidity to form freestanding 2D monolayers without substrates.Another important issue to confirm the stability of 2D materials is whether it can form a freestanding monolayer. Therefore, the in‐plane stiffness should be high enough to withstand its own weight or even external load without substrates. To check this, the in‐plane stiffness was calculated by −2) is comparable to the experimentally estimated value of graphite (≈0.37 J m−2),Ecl as shown in Figure 3  and MPSe3  (1.2–2.1 GPa) is even lower than that of graphite (≈2.1 GPa),Then the possibility of mechanical exfoliation from the bulk was checked by calculating the cleavage energy. The relative energy of the bulk increases with the separation distance and converges to the ideal cleavage energy gradually. The optimized lattice constants for the bulk structures are shown in Table S2 of the Supporting Information, which are consistent with the experimental data indicating the credibility of the calculations. As shown in Figure 3  and MPSe3  monolayers computed with Heyd–Scuseria–Ernzerhof (HSE06) functional are shown in Figure FigureWe next investigated the electronic properties of the monolayers. The band structures of MPS3 and MnPSe3 monolayers are direct‐band‐gap semiconductors with the band gap of 3.14 and 2.32 eV, respectively, consistent with the previous report.As shown in Table S3 of the Supporting Information, MnPS3 and MPSe3 monolayers, the partial density of states (PDOS) are computed. As shown in Figure p states of S/Se and the d states of metal atoms, respectively.To further understand the compositions of VBM and CBM for MPS3  and MPSe3  monolayers range from 1.90 to 3.44 eV, which exceed the free energy of water splitting of 1.23 eV. In addition to the magnitude of the band gap, the band edges must straddle the redox potentials of water. To check this, the work functions of these monolayers were calculated and are shown in Table S4 of the Supporting Information.The band structures and PDOS indicate that the band gaps of MPS+/H2 was calculated by EH+/H2red= −4.44 eV + pH × 0.059 eV and the oxidation potential for O2/H2O was calculated by EO2/H2Oox = −5.67 eV + pH × 0.059 eV. Considering that sulfides and selenides might be unstable in an acidic environment, the redox potential for water splitting reaction at neutral environment (pH = 7) was also calculated. The schematic diagram is shown in Figure3  and MPSe3  monolayers for photocatalytic water splitting. Except FePSe3, whose CBM is lower than the reduction potential of H+/H2 at pH = 7, the band edges of the rest compounds straddle the redox potentials of water at pH = 0 and 7. The results indicate that these materials are candidates for water‐splitting photocatalysts without an external bias voltage. More fascinatingly, besides the advantages of suitable positions of band edges at both acidic and neutral environment, MnPS3 and MnPSe3 are direct‐band‐gap semiconductors.For the water splitting reaction, the redox potential depends on the pH value.[[qv: 16b]],Another very important condition for photocatalytic water splitting is that the materials should capture a significant fraction of visible spectrum because it counts for more than 40% of the solar spectrum.Figureε2 of the dielectric function is shown in Figure S4 of the Supporting Information. The absorption coefficient is defined as the decay of light intensity spreading in a unit length of medium. FePSe3, MnPSe3, FePS3, and NiPS3 monolayers exhibit obvious optical absorption in visible spectrum and among them, the absorption of FePSe3 and MnPSe3 is stronger. However, FePSe3 might have no photocatalytic activity for water splitting at neutral environment. For MnPSe3, there are two absorption peaks in visible spectrum, indicating the strong optical absorption. Besides, the direct gap would make MnPSe3 more advantageous over other materials. The results indicate that MnPSe3 monolayer would exhibit better performance for photocatalytic water splitting. To investigate the effects of the layer number on the electronic properties and optical absorption of MnPSe3, MnPSe3 bilayer was considered. More details are shown in Figures S5–S7 of the Supporting Information. The results indicate that MnPSe3 bilayer could also exhibit catalytic activity for photocatalytic water splitting under visible light.To investigate the performance under light, the optical absorption coefficient was calculated and is shown in 3 monolayer as photocatalysts, the carrier effective mass and carrier mobility were investigated for quantitative evaluation on the ability to transfer electron/hole along the specific direction.To further evaluate the performance of MnPSeme*) and holes (mh*) are calculated by fitting parabolic functions to CBM and VBM, respectively. As shown in Tableme* and mh* along the direction of a  is shown in Table 2 V−1 S−1 while the hole mobility is 34.7 cm2 V−1 S−1. The carrier mobility of MnPSe3 monolayer could be comparable to or even higher than that of many other 2D semiconductors, such as MoS2 monolayer (≈200 cm2 V−1 S−1),2 V−1 S−1), fluorinated graphene (≈45 cm2 V−1 S−1), BN (≈487 cm2 V−1 S−1), and BC2N (≈180 cm2 V−1 S−1).To compute the DP constant 3 monolayer has many features, strong absorption in visible‐light spectrum, photocatalytic water splitting into H2 and O2 simultaneously, broad applicability (pH = 0–7), high carrier mobility, and feasible synthesis in experiments. Thus, MnPSe3 monolayer is a promising candidate as photocatalysts for water splitting.Overall, MnPSe3  and MPSe3  monolayers are promising candidates as photocatalysts for water splitting. Particularly, MnPSe3 monolayer is a direct‐band‐gap semiconductor which exhibits obvious absorption in visible‐light spectrum. Moreover, our calculations of effective mass and carrier mobility for MnPSe3 monolayer illustrate the transfer of carriers to the reactive sites would be easier and the probability of recombination would be lower for photogenerated carriers in the photocatalytic process. These results reflect that 2D MnPSe3 monolayer could be a promising photocatalyst for water splitting.In conclusion, the structure and stability of 2D metal phosphorus trichalcogenides monolayers were explored by first‐principles computations, which indicate that these 2D monolayers could be obtained in experiments by exfoliating the corresponding bulk. The calculated band gaps and band edge positions from accurate HSE06 functional predict that MPS3 sheets and the periodically repeated images. Moreover, considering that GGA functional systematically underestimates the band gaps,Our first‐principles computations based on density functional theory (DFT) were performed with a plan‐wave basis set as implemented in the Vienna ab initio simulation package (VASP).ε2 was calculated. The expression for ε2 was given as uck→is the cell periodic part of the orbital at the k‐point k→. The absorption coefficient α(ω) was calculated byε1 is the real part of dielectric function, which could be obtained from ε2 by Kramer–Kronig relationship.To investigate the optical absorption, the imaginary part of dielectric function T is the temperature, 300 K was adopted in this study, e is the electron charge, and h– is the reduced Planck constant. m* is the effect mass defined as m* = h–2(∂2E(k)/∂k2)−1, where k is the wave vector, and E(k) is the energy corresponding to the wave vector k. E1 is the DP constant denoting the shift of band edges induced by uniaxial strain, E1 = ∂Eedge/∂ε. C2D is the in‐plane stiffness as shown in Table S1 of the Supporting Information.For inorganic semiconductors, the electron coherence length is close to the acoustic phonon wavelength, which is much longer than the bonds. As a result, phonon scattering dominates the intrinsic mobility which can be described by the deformation potential theory.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.


Natural killer (NK) cells play a critical role in the innate immune response to viruses and tumors, and comprise a large proportion of the hepatic lymphocyte population. They must remain tolerant to non‐pathogenic antigens while protecting the host from harmful agents. Herein, we investigate how the NK cell response to activation receptor engagement is altered in the liver.In this study, we assess IFN‐γ production and degranulation of splenic NK cells and selected subsets of liver NK cells. Flow cytometry (FCM) was used to asses IFN‐γ production and degranulation following stimulation of the NK cells with plate bound antibodies to activating receptors.We show that smaller percentages of hepatic NK cells produce interferon (IFN)–γ and/or degranulate than do splenic NK cells upon stimulation through activating receptors. We also found that smaller percentages of the circulating NK (cNK) cells in the liver produce IFN‐γ and/or degranulate, compared to the liver tissue resident NK (trNK) cells. In addition, IFN‐γ production by liver cNK cells is not increased in IL‐10 deficient mice, suggesting that their hyporesponsiveness is not mediated by the presence of this anti‐inflammatory cytokine in the hepatic microenvironment. On the other hand, liver trNK cells express higher levels of the inhibitory receptor NKG2A than do cNK cells, correlating with their increased IFN‐γ production and degranulation.Liver cNK cells’ hyporesponsiveness to stimulation through activating receptors is independent of IL‐10, but correlates with decreased NKG2A expression compared to trNK cells. In addition, we demonstrate that liver NK cells become further hyporesponsive upon continuous engagement of an activating receptor on their cell surface. Natural killer (NK) cells play an important role in the innate immune response to a number of pathogens and are among the first cells to generate an immune response. They function by destroying abnormal cells through the secretion of cytotoxic granules, as well as secreting cytokines which shape the subsequent immune response Based on the mutually exclusive expression of two cell surface markers, NK cells within the liver can be divided into two populations. One population, tissue resident (tr) NK cells, express CD49a but not CD49b . The other population, conventional or circulating (c) NK cells, transit through the liver and express CD49b but not CD49a −, NKG2A+ population of NK cells described likely represents the trNK cells of the liver that have been described more recently Previous studies have suggested that liver NK cells are hyporesponsive compared to splenic NK cells based on their dampened IFN‐γ response to IL‐12/IL‐18 stimulation + liver NK cells to engagement of the NK1.1 activation receptor.In contrast to these prior studies, which assessed activation of NK cells using cytokines, in the present study, we sought to identify the factors involved in determining the response of liver NK cells to engagement of their activating receptors. We showed that smaller percentages of NK cells isolated from the liver produce IFN‐γ and/or degranulate when stimulated through activating receptors than do splenic NK cells, and that this functional alteration within the liver is limited to the cNK cells. In addition, IFN‐γ production by liver cNK upon stimulation cells is not increased in IL‐10 deficient mice, suggesting that IL‐10 does not mediate NK cell hyporesponsiveness within the hepatic microenvironment, as has been previously proposed. We found that increased IFN‐γ production and degranulation by trNK cells, compared to cNK cells, correlates with increased expression of the inhibitory receptor NKG2A. Finally, using an m157‐transgenic mouse model system, we demonstrated that continuous engagement of the Ly49H activating receptor results in a further dampened IFN‐γ response of Ly49HNK cells were harvested from the liver and spleen of wild‐type (WT) mice and assessed for the production of IFN‐γ following stimulation with plate‐bound antibodies to individual activating receptors (NK1.1 or Ly49D). When stimulated through NK1.1, a significantly lower percentage of liver NK cells produced IFN‐γ, compared to splenic NK cells  and cNK cells (CD49a−), we sought to determine if both or only one of these populations are hyporesponsive. To evaluate this, liver NK cells from WT mice were stimulated through NK1.1 and assessed for IFN‐γ production by the trNK cell and cNK cell populations. We found when stimulated through NK1.1, a significantly higher percentage of trNK cells produced IFN‐γ, compared to cNK cells  of NK cells from the liver and spleen in response to plate bound stimulation through NK1.1. We found that a significantly higher percentage of NK cells in the spleen underwent degranulation in response to this stimulation compared to liver NK cells Fig. A and B. We assessed the role that the anti‐inflammatory cytokine IL‐10, found in the liver microenvironment, might play in cNK cell function. Hepatic NK cells were harvested from IL‐10R2ko mice (in which the beta subunit of the IL‐10 receptor is not expressed), stimulated through NK1.1, and assessed for IFN‐γ production. IFN‐γ was produced by a significantly higher percentage of liver cNK as well as trNK cells from IL‐10R2ko mice compared to wild‐type mice Fig. A. SpleniIn addition to the IL‐10 receptor, the IL‐10R2 subunit is shared by the receptors for multiple interleukins, including IL‐22, IL‐26, IL‐28, and IL‐29 It has previously been demonstrated that inhibitory receptors can “license” or “educate” NK cells + and NKG2A− NK cells in both the spleen and liver following stimulation through the NK1.1 receptor. We observed that in both the spleen and the liver, NKG2A+ NK cells produced significantly more IFN‐γ compared to NKG2A− NK cells  and cNK cells , we still observed that a significantly higher percentage of NKG2A+ NK cells produced IFN‐γ as compared to NKG2A− NK cells  on NK cells in the liver microenvironment would further decrease the ability of NK cells to be activated via a separate activating receptor (NK1.1). To accomplish this, liver NK cells were harvested from both WT C57Bl6J mice and a transgenic (Tg) overexpressor (m157‐Tg) of the Ly49H receptor's ligand, m157. These were stimulated in vitro with plate‐bound antibody to NK1.1 and assessed for IFN‐γ production. The results are expressed as the ratio of the proportion of Ly49HNK cells are an important lymphocyte subset of the innate immune system. In addition to being found in the spleen and circulating in the blood, NK cells can be found in a number of solid organs. In fact, NK cells make up a large proportion of the hepatic lymphocyte population high cells were a precursor cells to NK1.1+, CD49b− NK cells residing in the liver (trNK cells) but not classical circulating NK cells (cNK cells) −, ID2+, IL‐7Rα+, CD25−, α4β7+, Flt3− precursor that gave rise to all helper‐like innate lymphoid cells  but not to cNK cells +, T‐bet+ while trNK cells are Eomes−, T‐bet+In addition to cell surface markers, several studies have demonstrated that tissue resident NK cells have different transcription factor requirements than circulating NK cells and likely arise from different progenitor cells The liver microenvironment provides a unique situation for the immune system in that it must be able to respond to harmful pathogens while at the same time remain tolerant to the many harmless antigens derived from commensal and food sources from the gut In this study, we demonstrate that hepatic NK cells produced significantly less IFN‐γ and degranulate less than NK cells from the spleen when stimulated through the engagement of cell surface activating receptors. In addition, NK cells from the hepatic microenvironment become further hyporesponsive (dampened IFN‐γ production upon stimulation of the NK1.1 activating receptor) when the Ly49H activating receptor is continuously engaged. This is similar to the activation‐receptor mediated hyporesponsiveness previously demonstrated in splenic NK cells − (corresponding to trNK cells) and Eomes+ (corresponding to cNK cells) NK cells within the liver. Although the Eomes− NK cells within the liver produced a wide variety of cytokines, they appeared to produce less IFN‐γ as compared Eomes+ NK cells upon stimulation through activating receptors + NK cells produced more IFN‐γ compared to CD49a− NK cells upon stimulation with PMA and ionomycin Previous studies have shown dampened response of hepatic NK cells to cytokine stimulation or treatment with PMA and ionomycin −) cells and not trNK cells (CD49a+) NK cells. In fact, our data suggest that a higher percentage of liver trNK cells produce more IFN‐γ and degranulate more when compared to liver cNK cells. In addition, a smaller percentage of cNK cells within the liver produced IFN‐γ and/or degranulate compared to cNK cells from the spleen suggesting that upon entering the liver from the circulation, cNK cells become hyporesponsive.Our studies revealed that the hyporesponsiveness of the total hepatic NK cell population is due to the presence of hyporesponsive cNK . Similar to previous work, we found that NKG2A levels are comparable on the splenic NK cells and liver cNK cells Studies assessing liver NK cells following adenoviral infection suggest that the CD49a expression may be difficult to use as a marker to differentiate cNK cells from trNK cells due to the fact that CD49a is transiently upregulated during activation of NK cells in vivo + NK cells, possibly providing a negative feedback mechanism NKG2A is a C‐type lectin‐like inhibitory receptor that interacts with the non‐classical MHC class I molecule Qa‐1b (human HLA‐E) The increased function of the liver trNK cells in the setting of elevated levels of the inhibitory receptor NKG2A on their cell surface seems counterintuitive and is in contrast to prior work suggesting that increased expression of NKG2A on liver NK cells is responsible for their hyporesponsiveness Several other studies corroborate the idea that NKG2A may in fact play a role in “educating” NK cells. It has been shown that NK cells which lack KIRs and NKG2A expression have decreased function and appear developmentally immature + NK cells within the liver that expressed a decreased number of inhibitory receptors Another study demonstrated that NK cells freshly isolated from surgical specimens from patients with hepatic malignancies have less cytolytic activity compared to those from autologous blood IL‐10 has been shown to have protective effects in multiple animal models of liver injury Similar to previous studies, we demonstrate that IL‐10 levels can alter NKG2A expression on NK cells Circulating NK cells from IL10R2 ko (knockout) mice (which do not express the IL‐10 receptor on their cells surface due to deletion of one of the subunits) displayed higher levels of IFN‐γ production as compared to WT mice upon engagement of activating receptors. However, cNK cells from IL‐10 ko mice did not produce more IFN‐γ as compared to WT mice upon engagement of activating receptors. Of note, the IL10R2 receptor subunit makes up a part of several receptors including those for IL‐22, IL‐26, IL‐28, and IL‐29 Several key differences could explain the discrepancies between our findings and those of prior studies. In our study, we assess IFN‐γ production and degranulation following stimulation with plate bound antibody to activating receptors rather than cytokine stimulation. In addition, we differentiated the NK cells into splenic, liver trNK and liver cNK, based on CD49a or CD49b expression, when assessing function. Finally, the mechanism in which the NK cells were isolated  from the liver likely plays a role in the ratio of trNK to cNK cells obtained, which could alter how the bulk NK cell population appears to function.It is now clear that NK cells have the ability to adapt to their environment and show plasticity in their response tm1AgtIl10rb/J) were obtained from Paul Allen and Tony French . The IL‐10ko mice (B6.129P2‐Il10tm1Cgn/J) were purchased from the Jackson Laboratory. Mice were maintained under specific pathogen‐free conditions and used after they reached 8 weeks of age. All animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86‐23 revised 1985). The Animal Studies Committee at Washington University approved all animal studies.The m157‐Tg mouse has been previously described The following antibodies were obtained from EBioscience  or Biolegend : APC or PerCPcy5.5‐PK136 (anti‐NK1.1), APC‐Cy7 or PerCP‐Cy5.5‐145‐2C11 (anti‐CD3), Alexa488‐XMG1.2 (anti‐IFN‐γ), APC or PE‐HMα1 (anti‐CD49a), Pacific Blue‐DX5 (anti CD49b), PE‐CD159a (anti‐NKG2A_B6), and streptavidin‐PE. The FITC‐16A11 (anti‐NKG2A) was obtained from Santa Cruz . The 3D10 (anti‐Ly49H) and 6H121 (anti‐m157) mAbs were purified from hybridomas by the Protein Production and Purification Core Facility of the Rheumatic Diseases Core Center at Washington University and conjugated to biotin using EZ‐Link Sulfo‐NHS‐LC‐LC‐Biotin  according to manufacturer's protocol. Purified PK136 (anti‐NK1.1) was purchased from BioXcell .g for 5 min. The supernatant was decanted and the pellet resuspended in 8 mL of R2  or R10  solution. The 8 mL of liver slurry was mixed with 5 mL of room temperature Percoll . Following adequate mixing, the solution was centrifuged at 835×g for 20 min at room temperature. The supernatant was removed and the remaining pellet was suspended in 10 mL of RBC lysis buffer for 5 min. Lysis was terminated by the addition of 5 mL of R2 or R10 solution, and the tube was centrifuged for 5 min. This pellet was washed with 10 mL of R2 or R10 solution and centrifuged again for 5 min. The resulting pellet of hepatic lymphocytes was resuspended in 1 mL of R10 solution.Livers were removed from mice and passed through a 100 μm filter. The slurry was then passed through a 70 μm filter and transferred into a 15 mL conical tube to be spun at 500×7 cells/mL in R10) or hepatic lymphocytes (approximately 1 × 107/mL in R10 for 96‐well plates and approximately 3 × 106/mL in R10 for 24‐well plates) were incubated in wells coated with anti‐NK1.1 or anti‐Ly49D mAb for 1 h and then further incubated in the presence of a 1000‐fold dilution of stock brefeldin A  for an additional 6–8 h. We used 40 μL of cells per well in the 96‐well plates and 333–500 μL of cells per well in the 24‐well plates. Cells were harvested and stained for surface markers, including NK1.1, CD3, CD49a, NKG2A, CD49b, and Ly49H. Cells were then fixed and permeabilized using Cytofix/Cytoperm solution , stained for IFN‐γ and analyzed on the FACSCalibur or FACSCanto .Splenic cells suspensions were generated as previously described NK cells were isolated from the liver and spleen and run through a Percoll gradient as described for the IFN‐γ assay. Liver or splenic NK cells were centrifuged onto 24‐well tissue culture plates that had been coated with 6 μg of PK136 for 2 h at 37°C. The NK cells were first stimulated for 1 h at 37°C in the presence of 5 μg/mL αCD107a‐FITC (BD Biosciences). Monensin (Biolegend) was added to achieve 2 μM, and the incubation continued for 5 additional hours. After the stimulation, cells were harvested and stained for surface markers, including NK1.1, CD3, CD49a, NKG2A, and CD49b. Cells were then analyzed by flow cytometry (FCM) on the FACSCanto flow cytometer.t‐tests or paired t‐tests were used to determine statistically significant differences between experimental groups. Error bars in the figures represent the standard error of the mean (SEM).The data were analyzed with Microsoft Excel . Unpaired, two‐tailed None declared.Additional supporting information may be found in the online version of this article at the publisher's web‐siteFigure S1. Tissue resident (tr) NK cells from WT mice express higher levels of NKG2A than trNK cells from IL10ko mice. The percentage of NK cells expressing NKG2A from WT  or IL10ko mice . NKG2A expression was assessed on splenic NK, liver cNK (CD49a+) and liver trNK (CD49a+) cells.Click here for additional data file.Figure S2. Liver trNK cells, but not cNK cells, differ from splenic NK cells in their expression of some inhibitory receptors. Unstimulated murine hepatic and splenic NK cells were stained for Ly49A, Ly49C, Ly49D, Ly49G2, Ly49H, Ly49I and NKG2A and evaluated by flow cytometry.Click here for additional data file.Figure S3. Stimulation with plate bound PK136 does not result in altered CD49a expression on liver NK cells.Click here for additional data file.


It is believed that the amorphous FePO4 layer could act as a lithium‐ions reservoir and electrochemically active buffer layer during the charge/discharge cycling, helping achieve high capacities in LiNi0.5Mn1.5O4, especially at high current densities. In regard of this, the majority of previous studies deposited ALD layer onto the surface of electrode sheet instead of material powders so as to avoid the insulation between binder, conductive carbon, and cathode materials since the coating on electrodes did not break the contact between them.+ within the voltage range of cathode materials are scarcely reported. With the electrochemically active advantage, direct coating onto powders becomes realistic.LiNiiMn2O4 14 mAh g–1.4 has been widely studied as cathode materials for both lithium‐ion batteries (LIBs) and sodium‐ion batteries (SIBs).4 via ALD process,–1.Amorphous FePO4 coating as a lithium‐ion reservoir during cycling, which may act as both a lithium diffusion facilitator and an electrochemical buffer layer between the electrolyte and LNMO by keeping the interface electrochemical potential above the electrolyte's highest occupied molecular orbital (HOMO), at which the electrolyte starts to get oxidized.4 coating based on X‐ray absorption spectroscopy (XAS) analysis.In this study, we propose the novel ALD‐derived ultrathin amorphous FePO4, no peaks of FePO4 can be observed in the XRD pattern. Figure−1 indicates that the LNMO is ordered P4332 phase, with subtle oxygen deficiencies. The peaks at 400 and 490 cm−1 are related to the Ni2+–O stretching and the peak at 630 cm−1 corresponds to the Mn–O stretching of MnO6 octahedra.4 cycles, both the Ni–O and Mn–O vibrations show blue shifts, this is due to the strains induced by the surface coating, which was also observed in TiO2 coating.p spectra were collected by synchrotron XPS technique. It can be observed that these P atoms on the LNMO surface show increasing concentration when the ALD cycle increases, indicating that the amount of surface coating layer correlates to the number of ALD cycles. It is worthwhile to note that the P 2p XPS spectra of LNMO‐40 did not show linear intensity increase, this is probably due to the surface saturation in synchrotron XPS.The preparation process of LNMO powders is described in the experimental section. The phases of LNMO were identified via XRD as shown in Figure S1 (Supporting Information). The peaks can be well indexed to the cubic spinel phase of LNMO (JCPDS No. 35–0782). ALD processes did not change the structures of the spinel LNMO. Due to the ultrathin and amorphous nature of the FePOn samples were characterized by FESEM , Figure Table4‐coated samples display increasing capacity retention with more ALD cycle numbers, indicating the protective nature of the FePO4 layer.4. Rate capability test , it can be seen that the Coulombic efficiency increases with the ALD cycle number, revealing that the presence of FePO4 has helped to suppress the electrolyte decomposition.n samples with normalized active material loading and electrolyte amount  show that the bare LNMO has much higher resident current value at the cutting voltage of 5.0 V than other samples, implying that the electrolyte oxidation in bare sample is more severe than coated samples. The lower area of the LNMO‐40 sample is also in accordance with the lower capacity. Table 4, suggesting that FePO4 coatings alleviates the polarization of the LNMO materials.Cyclic voltammetry (CV) measurements were carried out on the LNMO‐t Figure d. Three n sample after cycling for 100 times and subsequently charged to 5.0 V as shown in Figure4 coatings show two semicircles in the range of high and medium frequencies. A possible equivalent circuit is proposed to illustrate the impedance behaviors on the surface as shown in Figure RΩ stands for the Ohmic electrolyte resistance. The semicircle at high frequency is suggested to be a resistor Rs and a constant phase element (CPE), which are related to the migration of Li+ through the surface film, in this case, it reflects the resistance of SEI. Another semicircle at medium frequency is related to the charge transfer reaction composed of Rct and another CPE, together with the finite length Warburg impedance.Rs are presented in Table 4 coating, the Rs for LNMO‐0 is 173.1 Ω, however, the existence of FePO4 coating layer helped to decrease the Rs values dramatically, which vary monotonically with the number of ALD cycles, to only 57.9 Ω for the LNMO‐40 sample. The drop of Rs clearly reveals the less formation of insulating SEI, which is a result of electrolyte decomposition, therefore FePO4 is effective towards suppressing the electrolyte decomposition.In the effort to understand the formation of solid electrolyte interface (SEI) on the surface of the electrodes, AC electrochemical impedance spectra (EIS) were conducted on each LNMO‐n samples, XANES was collected on the Mn L3,2‐edges. Mn L3,2‐edges illustrate the electronic transition from Mn 2p3/2 and 2p1/2 to an unoccupied 3d state.Figure2‐edge often appears to be broader due to the core hole lifetime as explained by Coster–Kronig Auger decay.4+ features that fit well with standard MnO2, the small peak at 646 eV corresponds to Mn3+, and this is also consistent with the Raman spectra, the unchanged spectra reveal that the coating process did not generate changes to the surface phase of LNMO. However, after charge/discharge cycling, Mn4+ at the surface was partially reduced to Mn2+, and the LNMO‐0 shows much higher intensity ratio of Mn2+/Mn4+ than the coated LNMO sample. The bulk‐sensitive fluorescence yield (FYI) spectra of LNMO‐20, LNMO‐20 after battery cycling and LNMO‐0 after battery cycling are shown in Figure S8 (Supporting Information). It can be seen that the bulk Mn exhibits subtle changes after cycling. The less reduced Mn valence on coated LNMO surface also reveals weaker reduction by the electrolyte, which can be attributed to the inhibitive role of FePO4 against the electrolyte oxidation.3+ triggers the Jahn–Teller distortion because of its (2gt3–ge1) configuration, resulting in its charge disproportionation into non Jahn–Teller active Mn2+ and Mn4+, described as 2Mn3+ → Mn2+ + Mn4+.[[qv: 3a]],6 salt, Mn2+ ions dissolve in the electrolyte and migrate through the separator followed by depositing on the anode as Mn metal, with a secondary phase formed on the surface of cathode materials.4 coating prevents the formation of Mn2+, thereby decreases the chance of Mn2+ dissolution in HF, and improves the stability of LNMO.To investigate the change of Mn valence state in the LNMO‐3‐edges XANES of standard FePO4, LNMO‐20, and LNMO‐20 after battery cycling were performed to determine the chemical states of the FePO4 coatings before and after charge/discharge cycling. As shown in Figure 4 spectrum, the intense peak at 713.5 eV  and the weaker peak at 712.2 eV are related to the spin‐orbit, interplay of crystal‐filed, and electronic interactions. Their intensity ratio reveals the Fe3+/Fe2+ ratio.3+/Fe2+ ratio, indicating that part of the Fe3+ has been reduced, and the position of the right peak is, interestingly, shifting to higher energy value. Such shift was also observed in our previous study on the soft XANES spectroscopies of FePO4‐related various phases.4 has resulted in the partially lithiated FePO4 domains, which acts as a lithium‐ion reservoir and exhibited improved performance at high current densities by providing abundant Li+ diffusion pathways.Fe L4 is presented in Figure4 coating is resistant to the side reactions. This is because it was found that the noncoated sample displayed Mn at reduced state on the surface after cycling, which is much more prone to dissolution compared with Mn3+ and/or Mn4+.[[qv: 29b]] Additionally, the amorphous FePO4 layer accommodates lithium ions rapidly during cycling, thus provides fast lithium diffusion. More specific role of FePO4 is shown in Figure 4 and LiNi0.5Mn1.5O4. The electrolyte gets readily oxidized when the electrochemical potentials of cathode materials are below the HOMO of it.2O3 or ZrO2, and FePO4 is electrochemically active with an open‐circuit voltage of ≈3 V,[[qv: 15a]] the FePO4 ultrathin layer on the surface prevents the direct contact of LNMO with the electrolyte, helping to avoid the oxidation of electrolyte that results in the reduction and dissolution of Mn ions.Based on the aforementioned results, the schematic illustration of the protecting role of FePO4 coating on high voltage LNMO cathode material enabled by ALD. Different thicknesses of FePO4 have been deposited onto LNMO powders with 5, 10, 20, and 40 ALD cycles. The LNMO coated with 10 ALD cycles of FePO4 showed the best performance including the highest capacity and stabilized capacity retention under all the current rates. When the LNMO was coated with 40 ALD cycles of FePO4, the capacity retention increased up to 100%. XANES study showed that the ultrathin FePO4 suppressed the surface Mn4+ from being heavily reduced to Mn2+ by the reduction from the electrolyte and the Jahn–Teller distortion, less amount of Mn2+ helped to retain the surface consistency without severe dissolution into the electrolyte. The FePO4 coating layer was slightly reduced due to the remaining Li+ in the structure after charge/discharge cycling. Compared with the most widely used insulating Al2O3, amorphous FePO4 presents many advantages on the electron/ion diffusion on the surface. Our work provides an alternative option of depositing materials onto powders instead of electrode sheets directly using ALD, which expands the deposition temperature, owing to the electrochemically active nature of FePO4.We have proposed a new FePOMaterials Synthesis: LiNi0.5Mn1.5O4 was synthesized via a two‐step hydrothermal‐assisted carbonate precipitation method followed by thermal treatment. Ni(NO3)2·6H2O  and Mn(NO3)2· 4H2O  were dissolved in deionized water (5 mL). Na2CO3  solution was subsequently added to the above mixture of nickel nitrate and manganese nitrate under vigorous stirring at a rate of 0.25 mL min–1, then the green precipitation was transferred to a 40 mL Teflon‐lined autoclave and kept at 140 °C for 10 h. After cooling down to room temperature (RT), the precipitation was filtered and washed with water several times and dried at 80 °C overnight. The carbonate powders were annealed at 450 °C for 4 h in air so as to obtain corresponding oxides. Thereafter, the oxide powders were mixed with Li2CO3  in 1:1 water and ethanol mixture (10 mL) and left to dry under stirring at 60 °C. The mixed precursor was subsequently sintered in O2 at 800 °C for 6 h and then cooled to 600 °C in 3 h. After keeping at 600 °C for another 6 h, the furnace was cooled to RT at a cooling rate of 1 °C min−1 to obtain the final LNMO.4 was deposited at 300 °C by using ferrocene , ozone , trimethyl phosphate 3PO, 97% STREM Chemicals), and distilled water (H2O) as precursors in a Savannah 100 ALD system . The source temperature for FeCp2 and TMPO was 130 and 75 °C respectively. O3 and H2O were fed into the reactor chamber at RT. The deposition of FePO4 was achieved by following a sequence of FeCp2 pulse (1 s) – purge (10 s) – O3 pulse (1 s) – purge (10 s) – TMPO pulse (2 s) – purge (10 s) – purge (10 s) – H2O pulse (1 s) – purge (10 s). Nitrogen gas (99.999%) was used as a carrying and purging gas at a flow rate of 20 sccm. The above processes were repeated for several (n) times to grow n cycles of FePO4 onto LNMO powders, denoted as LNMO‐n (bare LNMO when n = 0).Amorphous FePOCharacterization Methods: The morphology of LNMO‐n was characterized by a Hitachi S‐4800 field emission scanning electronic microscopy (FESEM) equipped with an energy dispersive X‐ray spectroscope (EDS), Hitachi H‐7000 transmission electron microscope (TEM), and a high‐resolution transmission electron microscope . Raman scattering (RS) spectra was collected from a HORIBA Scientific LabRAM HR Raman spectrometer system with a 532.4 nm laser and optical microscope at RT. X‐ray diffraction (XRD) patterns were collected on a Bruker D8 Advance Diffractometer using Cu Kα radiation at 40 kV and 40 mA. The X‐ray absorption near edge structure (XANES) measurements at total electron yield (TEY) and fluorescence yield (FYI) modes of Mn L2,3‐edge and Fe L3‐edge were performed at the Canadian Light Source (CLS) on the high resolution Spherical Grating Monochrometer (SGM) beamline using a 45 mm planar undulator and three gratings with a photon energy range of 250–2000 eV, LNMO‐20 was chosen as the target sample. The P 2p X‐ray photoemission spectroscopy (XPS) was performed at the variable line spacing plane grating monochromator (VLS PGM) beamline at 200 eV photon energy with a total resolution of 100 meV.Electrochemical Measurements: The LNMO‐n powders were mixed with poly(vinylidene fluoride) binder and acetylene black in a ratio of 8:1:1 in N‐methyl‐pyrrolidione (NMP) solvent to form slurries. The slurries were subsequently casted onto aluminum foils as the current collector and dried at 80 °C under vacuum overnight. The electrode was assembled in an Ar‐filled glovebox with moisture and oxygen concentrations below 1 ppm. A CR‐2032 type coin cell using a lithium metal as the counter electrode and Celgard K2045 as the separator was utilized. The electrolyte was composed of 1 m LiPF6 salt dissolved in ethylene carbonate (EC) and dimethyl carbonate (DMC) in a 1:1 volume ratio (BASF Corp.). Cyclic voltammetry (CV) was performed on a versatile multichannel potentiostat 3/Z (VMP3), with a scanning rate of 0.1 mV s−1 and a potential range of 3.5−5.0 V (vs Li+/Li) at RT. Electrochemical impedance spectroscopy (EIS) was also performed on the versatile multichannel potentiostat 3/Z (VMP3) by holding the cells at 5.0 V. Galvanostatical charge/discharge was performed on Arbin BT2000 at various current densities between 3.5 and 5.0 V (vs Li+/Li), the stability performance test was done under 0.5 C, which is 73.5 mA g–1.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.


Top2β, phrenic MN identity is eroded, while other motor columns are partially preserved but fail to cluster to their proper position. In Top2β-/- mice, peripheral connectivity is impaired as MNs exhibit a profound deficit in terminal branching. These defects likely result from the insufficient activation of Hox/Pbx-dependent transcriptional programs as Hox and Pbx genes are downregulated in the absence of Top2β. Top2β mutants recapitulate many aspects of Pbx mutant mice, such as MN disorganization and defects in medial motor column (MMC) specification. Our findings indicate that Top2β, a gene implicated in neurodevelopmental diseases such as autism spectrum disorders, plays a critical, cell-specific role in the assembly of motor circuits.Vital motor functions, such as respiration and locomotion, rely on the ability of spinal motor neurons (MNs) to acquire stereotypical positions in the ventral spinal cord and to project with high precision to their peripheral targets. These key properties of MNs emerge during development through transcriptional programs that dictate their subtype identity and connectivity; however, the molecular mechanisms that establish the transcriptional landscape necessary for MN specification are not fully understood. Here, we show that the enzyme topoisomerase IIβ (Top2β) controls MN migration and connectivity. Surprisingly, Top2β is not required for MN generation or survival but has a selective role in columnar specification. In the absence of  The acquisition of motor neuron (MN) identity is a critical step in the assembly of motor circuits but the molecular pathways underlying MN specification remain unclear. Here, we show that the enzyme topoisomerase IIβ (Top2β) differentially controls MN subtype specification. In the absence of Top2β phrenic MNs do not develop and mice die due to respiratory failure, while other MN subtypes are partially preserved, demonstrating a phrenic-specific critical role for Top2β. We show that Top2β acts via Hox/Pbx-dependent transcriptional programs to control MN organization and medial motor column (MMC) specification, thus unraveling a key step in the genetic hierarchy that underlies MN development.in vitro, have nonoverlapping functions. Topoisomerase IIα (Top2α) is required for cell viability and chromosome segregation while Top2β is dispensable for basic cellular functions and appears to act predominantly within the nervous system  neurons  class of homeodomain proteins, act as cofactors for Hox proteins to establish high affinity binding to transcriptional targets  neurons and their guidance to the diaphragm. The establishment of PMC identity relies on the activity of Hox5 transcription factors (TFs) at cervical levels of the spinal cord . Hox pro neurons . While i targets . In MNs,pography .Hox gene expression in the spinal cord are initially established though morphogen gradients and maintained through Polycomb-dependent repression mechanisms  specification. Our results demonstrate that Top2β regulates MN identity in a cell-specific manner by ensuring robust activation of Hox/Pbx-dependent transcriptional programs.To understand how Top2β controls NMJ formation and at which level of the transcriptional hierarchy that underlies MN development it acts, we assessed its role in MN specification and peripheral connectivity. Surprisingly, Top2β-/- (Hb9::GFP (The Top2β-/-  and Hb9:In situ hybridization and immunohistochemistry were performed as described (AB_2205866), rabbit anti-cleaved Caspase 3 , rabbit anti-Pbx1 , mouse anti-olig2-A488-conjugated , rabbit anti-GFP , goat anti-Scip , rabbit anti-Neurofilament , rabbit anti-Mecom , and a-bungarotoxin, Alexa Fluor 555 conjugate  . Images were obtained with a Zeiss (LSM 800) confocal microscope or a Zeiss Imager Z2 epifluorescent microscope with Apotome and analyzed with Zen Blue and ImageJ (Fiji).escribed . Wholemoescribed  and motoescribed . Antibodescribed . Other at test.For all experiments a minimum of three embryos per genotype, both male and female, were used for all reported results. In experiments where MN numbers are reported, cells were counted as MNs if they expressed Isl1/2 and as LMC neurons if they expressed FoxP1. Counts were performed in the rostral brachial spinal cord, at the same rostrocaudal levels for both control and mutant animals. The MN numbers reported are either the average  or the total (Scip+) of three to four consecutive sections separated by 160 μm. For the quantitation of Scip, FoxP1, Hox, and Pbx protein levels, the fluorescence intensity in MNs in the ventral spinal cord along three to four consecutive sections was measured in ImageJ (Fiji) and the average fluorescence for each protein was then calculated. Average fluorescent intensity for control protein levels was set to 1, and protein levels in the mutant were expressed as a fraction of control levels. P-values were calculated using paired, two-tailed Student’s + MN progenitor domain and increases as MNs differentiate and move away from the ventricular zone  at multiple embryonic stages and we did not observe a reduction in MN numbers in Top2β-/- mice (Vacht), as detected by in situ hybridization, also revealed similar numbers of cholinergic MNs in control and Top2β mutant mice  and latissimus dorsi (LD) muscles express the TF Pea3. In Top2β mutant mice both Scip and Pea3 are dramatically downregulated and the remaining MNs expressing either TF are no longer clustered  synthetic enzyme nal cord F. This flustered G,H. Our Top2β mutation on MN axonal trajectories, we examined limb innervation by wholemount immunofluorescence. In FoxP1-/- mice, peripheral patterns of innervation are preserved despite the erosion of MN identity and the absence of pool marker expression  indicating that the three major limb-innervating motor nerves are impacted in a similar manner. The increase in nerve thickness is likely a result of a rerouting of all MNs along these 3 major tracts. Consistent with this idea we observe a loss of proximal motor nerves, such as the axillary and lateral pectoral nerves, in Top2β-/- mice  are defined by low levels of FoxP1 and neuronal nitric oxide synthase (nNOS) expression . Despite a decrease in the expression of PGC markers and displacement of their cell bodies in erminals D. These Top2β affects Hox and Hox cofactor protein expression. We first assessed the expression of Hox proteins known to have an instrumental role in MN columnar specification: Hoxa5 for PMC, Hoxc6, and Hoxc8 for brachial LMC and Hoxc9 for PGC. In the absence of Top2β, the rostrocaudal boundaries of Hox protein expression are preserved, indicating that MN defects are not due to altered Hox protein spatial distribution (Figs. 7E–H, 8G; Top2β-/- mice. Surprisingly, levels of Hoxa5, the major Hox determinant of PMC identity, were unchanged indicating that the absence of phrenic MNs is not a result of decreased Hoxa5 activity and that Top2β potentially regulates additional PMC determinants.How does Top2β activity contribute to the subtype specification of spinal MNs? Since the acquisition of both columnar and pool identities requires the activity of Hox TFs along the rostrocaudal axis, we examined whether removal of ribution A–D. We dTop2β mutants, we find that levels of both Pbx1 and Pbx3 are greatly reduced at e11.5 and this loss was observed at all rostrocaudal levels of the spinal cord (Pbx genes function as downstream effectors of Top2β and that their loss may be responsible for the phenotypes observed in Top2β-/- mice.Pbx1 and Pbx3 are critical cofactors for Hox proteins and are required for PMC specification . In Top2nal cord A–G. ThisTop2β mutants exhibit additional defects in MN specification that are similar to those observed in Pbx mutants. While Pbx genes are required for the induction of Hox-dependent MN specification programs, they also have independent roles in MN topography and axial muscle-innervating MMC neuron development. We examined whether MNs in Top2β mutants exhibit similar disorganization as Pbx mutants. Unlike in control embryos, where MNs expressing Isl1/2 and Hb9 progressively segregate, we found that these MN populations were intermixed both at brachial and thoracic levels in Top2β-/- mice (Top2β-/- embryos, Isl1/2+ MNs occupied a more medial position in the spinal cord and failed to migrate to their most dorsal positions, similar to the migration defects observed in Pbx mutants (To test whether Pbx proteins act downstream of Top2β, we examined whether -/- mice A–D. In T mutants .Top2β-/- mice. MMC neurons are found at all rostrocaudal levels of the spinal cord and their development is thought to be Hox-independent. However, MMC-restricted genes are depleted in Pbx mutants, indicating that Pbx genes play a Hox-independent role in the specification of this population (Mecom and lifr, two genes that require Pbx proteins for MMC-restricted expression. Both of these genes were found to be downregulated in Top2β mutants (Top2β mutants. Both the TF Lhx3 and the cytokine receptor gp130, which are highly enriched in the MMC, persist in Top2β mutants (We next examined whether MMC specification is affected in pulation . We exam mutants E,F. It i mutants . Our resThe acquisition of proper MN subtype identity is a critical step in the assembly of motor circuits and the execution of vital functions such as respiration and locomotion. We found that Top2β plays an essential role in MN development, acting through early, subtype-specific transcriptional programs during MN columnar and pool differentiation. We discuss the role of Top2β in MN development and potential mechanisms of Top2β action.Top2β mutants (Top2β mice die at birth from respiratory dysfunction due to the lack of synapses at the diaphragm but the mechanisms by which Top2β affects NMJ formation and the exact function of Top2β in MNs had not been resolved (Top2β-/- phrenic MNs are not impaired in their ability to form synapses as previously thought and that Top2β acts early in postmitotic PMC neurons to establish specification programs. Our study points to potential mechanisms for Top2β involvement in neurodevelopmental disorders, not necessarily at the time of synapse formation but rather early in development as neuronal subtypes are being generated.Type II topoisomerases solve DNA topological problems by transiently creating double strand breaks to relieve torsional stress during DNA processes such as replication and transcription. Top2β has been implicated in nervous system development and multiple neuronal populations are impaired in  mutants . In the resolved . We demoTop2β in vivo was shown to affect survival and neurite outgrowth in multiple classes of neurons, suggesting that Top2β may control generic pathways active in multiple neuronal populations to promote axon growth and survival (Top2β is not required in vivo for MN survival. We do not observe an upregulation of p75 and a resulting increase in apoptosis as has been reported for other neuronal populations, indicating that Top2β plays distinct roles in different neuronal subtypes. In Top2β mutants MNs extend their axons to the periphery and grow along major tracts in the limb but subsequently fail to branch to their specific muscles. These defects in peripheral projections appear to be specific for certain classes of MNs; projections along the phrenic nerve are dramatically reduced while LMC neurons project along major nerve tracks in the limb but fail to branch to individual muscles. This specificity argues against a generic function of Top2β in neurite outgrowth, we rather favor the idea that Top2β acts during a critical window in early MN development to orchestrate the specification of MN subtypes. Our results highlight an unexpected specificity for Top2β function and offer support to the idea that Top2β disruption in the nervous system can have subtle, specific effects, manifesting for example as autism spectrum disorders (Pharmacological inhibition of Top2β in cultured neurons and deletion of survival . Our datisorders , rather Top2β different MN columns are impacted with varying severity (Top2β mutants do not show induction of any phrenic-specific markers, few axons project along the phrenic nerve and diaphragm innervation is completely absent. While LMC and PGC neurons also appear disorganized, several column-specific markers, such as FoxP1 and nNOS, respectively, are partially preserved. The complete erosion of PMC identity reveals an increased sensitivity of the phrenic MN population to Top2β deletion, suggesting the specific regulation of PMC determinants by Top2β. PMC identity is dictated by select transcriptions factors including Hox5 proteins and their cofactors Pbx1 and Pbx3 (Pbx mutants display defects in PMC specification and diaphragm innervation that are similar to that of Top2β mutants.Our results indicate that Top2β has a critical role in the specification of MN columnar identity and in the absence of severity . Phrenicand Pbx3 . Our datPbx1/3 and Top2β mutants exhibit similar phenotypes in regards to phrenic MNs, the peripheral projections of LMC neurons are differentially affected, with Top2β mutants showing increased projections along major nerves while Pbx1/3 mutants exhibit thinning of most limb-innervating axons. In Top2β mutants FoxP1 and Pbx expression is partially preserved, compared to a complete absence in Pbx1/3 mutants. This difference in the levels of FoxP1 and Pbx likely contributes to the differential effects on LMC connectivity observed. Our data also reveal an increased sensitivity of PMC neurons to fluctuating Pbx levels and a potential dependence of these neurons on high Pbx expression. Both Pbx1 and Pbx3 show highest expression in the cervical spinal cord where phrenic MNs are located when compared to the rest of the spinal cord, suggesting that high levels of the two factors are required in this population. A reduction of Pbx levels by 50% is sufficient to prevent the induction of all PMC markers but only partially affects LMC and PGC induction. Our data underscores that in addition to the temporal and spatial restriction of TF expression, robustness of expression, an often-underappreciated dimension of developmental programs, is also a significant determinant of MN identity.While Hox and Pbx genes and impact on early MN specification programs, the deletion of Top2β may also influence the expression of a cohort of MN specific genes involved in later aspects of development such as synaptogenesis. A screen for genes downregulated in cortical neurons in Top2β mutants at multiple time points revealed a high number of affected genes, raising the possibility that Top2β may have a more global role in transcription throughout development (In addition to downregulation of elopment . It is aTop2β mutant mice, suggesting that a common set of genes is under Top2β regulation that can influence different functions depending on the neuronal cell type (Top2β-/- brain, suggesting that the dramatic change we observe likely results from a defect in PMC specification rather than a direct effect of Top2β on ALCAM transcription. Consistent with this idea a number of genes that are affected in Top2β mutant brains do not exhibit Top2β binding at their promoter regions, indicating that some expression changes are an indirect result of TF downregulation. It has been suggested that the transcription of long genes, including autism-spectrum disorder risk genes, is disproportionately affected in the absence of Top2β activity (Top2β mutants and distinguishing between Top2β direct and indirect targets in MNs will further illuminate the role of this protein in MN development.Interestingly, a microarray screen in the embryonic brain identified Pbx1 and Pbx3 as being downregulated in ell type . The PMCactivity . Pbx1 anTop2β mutants result from a reduction in the expression of key TFs that act during early MN development. Our findings highlight the importance of titrating TF levels during neuronal specification. In MNs for example, robust expression of FoxP1 leads to the acquisition of LMC identity, while low levels of FoxP1 underlie PGC specification (Top2β mutants indicate that certain MN subtypes are more sensitive to even small fluctuations in TF expression (Top2β-/- mice indicating that there is a critical requirement for high Pbx expression in the induction of PMC-specific genes. In contrast, LMC-specific Raldh2 expression, which is abolished in Pbx1/3 mutants, persists in Top2β mutants, indicating a less stringent requirement for high Pbx levels for the expression of LMC determinants. Our data suggests that the degree of robustness of transcriptional regulatory networks, established through the activity of Top2β, is a critical determinant of neuronal cell identity programs and serves as an additional strategy employed in the nervous system to generate cell diversity. An intriguing possibility is that Top2β also acts in multiple interneuron populations in the spinal cord to initiate further subtype specification. In support of this idea, Top2β-/- mice show changes in the localization of Lhx3+ interneurons in the spinal cord (Top2β as a critical mediator in the assembly of motor circuits and Top2β mutant mice will provide a powerful tool to further dissect the transcriptional pathways that give rise to diverse populations of neurons in the mammalian spinal cord.The complex phenotypes observed in fication . The difpression . The spenal cord H. Our fi