The ordering of cations and oxygen vacancies is discussedin comparison with the related Li2CuO2□1 and Li5SbO5□1 phases.The electrochemical properties of this material are presented, withonly 0.55 Li+ extracted upon oxidation, corresponding toa limited utilization of cationic and/or anionic redox, whereas morethan 2 Li+ ions can be reversibly inserted upon reductionto 1 V vs Li+/Li, a large capacity attributed to a conversionreaction and the reduction of Cu2+ to Cu0. Controlof the formation of oxygen vacancies in Li-rich rocksalt oxides byselecting appropriate cations and synthesis conditions affords a newroute for tuning the electrochemical properties of cathode materialsfor Li-ion batteries. Furthermore, the development of material modelsof the required level of detail to predict phase diagrams and electrochemicalproperties, including oxygen release in Li-rich rocksalt oxides, stillrelies on the accurate prediction of crystal structures. Experimentalidentification of new accessible structure types stabilized by oxygenvacancies represents a valuable step forward in the development ofpredictive models.Li-rich rocksaltoxides are promising candidates as high-energydensity cathode materials for next-generation Li-ion batteries becausethey present extremely diverse structures and compositions. Most reportedmaterials in this family contain as many cations as anions, a characteristicof the ideal cubic closed-packed rocksalt composition. In this work,a new rocksalt-derived structure type is stabilized by selecting divalentCu and pentavalent Sb cations to favor the formation of oxygen vacanciesduring synthesis. The structure and composition of the oxygen-deficientLi 4CuSbO5.5 is described and discussed in the context ofother oxygen-deficient rocksalt oxides.Controllingthe composition and synthesis conditions ofLi-rich rocksalt oxides can lead to to new structure types stabilizedby oxygen vacancies. The complex atomic ordering of Li However, this increase in capacity is oftenaccompanied by several shortcomings linked to stabilization mechanismsfor oxide anions upon their oxidation. Common issues range from voltagehysteresis between charge and discharge, resulting in a low-energyefficiency, to cation migration and trapping in tetrahedral sites,leading to voltage decay, and irreversible evolution of oxygen fromthe surface and/or the bulk of the material that causes irreversiblecapacity loss.1 Research efforts are thereforefocused on understanding how the redox activity of the anion sublatticecan be controlled and finely tuned through appropriate choice of cations,including redox active transition metals and redox inactive elements.The development of high-energy density cathode materials for Li-ionbatteries has opened a large field of investigation for solid-statechemists to propose new materials and new energy storage concepts.The discovery of the reversible redox activity of oxygen anions inthe so-called “Li-rich” rocksalt oxides with formulaLi5 or complete amorphization in the most extreme cases.6 Computational evaluation of the propensity for a specificcomposition to undergo oxygen release and oxygen vacancy formationrepresents a promising path to guide future experimental explorationof high-energy density cathode materials. Several research groupshave already taken that direction,8 reporting different indicators to predict the releaseof oxygen and leading to a more general understanding of the reversibilityof oxygen redox activity. Nevertheless, these indicators rely on theaccurate prediction of the structure formed upon the introductionof an oxygen vacancy and would benefit from the knowledge of experimentallydetermined crystal structures adopted by Li-rich rocksalt oxides containingoxygen vacancies.The release of gaseous oxygen is a particularlyimportant problem,as it results in irreversible capacity loss and penalizing structuralmodifications such as densification of the surface of materials4MM′Ox6–□x, where M and M′ cations must be selected in order to favorthe formation of oxygen vacancies during synthesis rather than duringelectrochemical oxidation.In this work, the formation of structureswith oxygen vacanciesis taken as an opportunity to discover new structure types relatedto the Li-rich rocksalt oxides, with formula Li10 This mechanism of reductive elimination is an extreme and irreversiblemanifestation of the reductive coupling mechanism observed in somecathode materials.2 To achieve this reactionat the synthesis step, we further replaced Ni/Fe with Cu, which isunlikely to oxidize beyond divalent Cu2+ under normal solid-statesynthesis conditions . Therefore,we targeted the composition Li4CuSbO5.5□0.5 and report its structure and electrochemical properties.In this new compound, the sum of M and M′ cation oxidationstates amounts to 7 instead of 8, as is usually considered to preparestoichiometric Li4MM′O6 rocksalt oxides.Work from McCalla and co-workerson Li–Fe–Sb–Oand Li–Ni–Sb–O compounds suggests that Sb-basedmaterials are prone to oxygen release from the bulk or the surfaceof the material, together with cation reduction upon deep oxidation.22.14CuSbO5.5 can be preparedby a simple ceramicmethod starting from Li2CO3, CuO and Sb2O3 in proportions Li:Cu:Sb = 4.4:1:1 and heatingthe hand-ground mixture at 1100 °C for 12 h under air  to be Li4.22(9)Cu1.027(18)Sb1.000(2), with deviationsfrom the ideal stoichiometry that can be explained by residual Li2CO3/Li2O, Li2CuO2 and Li7SbO6 impurities .12Litructure 1a, greenO5 phase 1a, gray.a = 5.207365(8) Å, b = 5.817536(8) Å, c = 7.888147(13)Å, α = 100.58195(11)°, β = 96.93693(11)°,and γ = 106.96091(9)°. All attempts to index the patternwith a higher symmetry cell were unsuccessful. Initial positions forSb, Cu, and O atoms were found by simulated annealing using the programFOX13 and further determined through Fourierdifference maps as implemented in the FullProf suite.14 Li positions were determined from Fourier difference mapsusing neutron diffraction (ND) data. Overall site occupancies andatomic displacement parameters were determined by a Rietveld refinementcombining synchrotron X-ray diffraction (SXRD) data with neutron diffractiondata (1) lattice plane (sliceshown at x = 0) clearly support the absence of oxygenin site 1b  Å, b = 5.82002(4) Å, c = 7.88000(6) Å, α= 100.4694(6)°, β = 97.0145(5)°, γ = 107.0248(6)°)improved the fit significantly  pattern of the sample prepared at 1100 °C for 12 h usingsynchrotron radiation. The structure can be indexed using a tricliniccell with cell parameters: iondata 1b. Thecionssee  section.2.24CuSbO5.5 is derived from the cubic rocksaltstructure of NaCl  latticeplane (b). The cation ordering is closely related to those of theparent compounds Li2CuO2  and Li5SbO5 (or Li4LiSbO5□1), as shown in 4(MM′)Ox6–□x: Li4(CuCu)O4□2, Li4(LiSb)O5□1 and Li4(CuSb)O5.5□0.5 with decreasing vacancy content, respectively. The number of oxygenvacancies x is directly related to the sum of oxidationstates m and m′ of the cationsM and M′ (x = (8 – m – m′)/2).The structureof Li of NaCl 2a–c. Theceplane 2d,whichceplane 2a. This Immm cell of Li2CuO2, the (100) planein the monoclinic C2/m cell of Li5SbO5, and the (101) planein the triclinic cell of Li4CuSbO5.5. The Mand M′ sites share edges along one direction  and corners in the perpendiculardirection . Layersof Li atoms separate each (MM′) layer from the next one (4(CuCu)O4□2, all M and M′sites are occupied by Cu2+, so that the oxygen corner-sharingsites remain empty, resulting in a square planar coordination forall Cu atoms O5□1, the highvalence of Sb5+ cations prevents oxygen vacancies fromforming in their immediate surrounding, preserving the octahedralcoordination environment. Instead, oxygen vacancies are found betweenLi+ ions, which are, therefore, sitting in a square planarenvironment. Sb sites form chains of corner-shared octahedra alongthe [010] direction, and each chain is separated from the next oneby square planar Li sites in the [001] direction (4(CuSb)O5.5□0.5, we now understandthat oxygen vacancies will preferentially be found in corner-sharedsites between two Cu2+ cations, rather than at a vertexof the octahedral Sb5+ cation site. This is possible byordering Cu2+ and Sb5+ cations in the (101) plane to alternate between Cu and Sb along the edge-sharing[010] direction −(Cu–Sb–Cu–Sb)–and between pairs of Cu and Sb along the corner shared [212] direction−(Cu–Cu–Sb–Sb)–  = 2.288(5) Å)is significantly longer than the Cu–O equatorial distancesforming the base of the pyramid (1.975(6)–2.045(7) Å)due to the strong Jahn–Teller distortion discussed above . The Sb5+ cations reside in pairs of octahedralsites connected by a corner. Generally, the repulsion between highlycharged cations in rocksalt oxides is the main driving force for cationordering, leading to completely isolated MO6 octahedrato minimize electrostatic energy. However, the Sb2O11 dimers present in Li4(CuSb)O5.5 indicatethat cationic repulsion comes only second to spreading oxygen vacanciesin the structure. The SbO6 octahedra show little deviationfrom a perfectly regular octahedral environment except for a slightdisplacement of Sb5+ cations away from each other (d(Sb8–O6) = 2.0307(8) Å > d(Sb8–O5) = 1.991(5) Å). This displacement is likely theresult of the minimization of electrostatic energy between the twohighly charged Sb5+ cations. It also helps to mitigatethe deviation from an ideal bond valence sum (BVS) of two for thecorner-sharing oxygen (O6) at the apex of the two Sb octahedra. Thisoxygen site is coordinated to two Sb5+ and four Li+ cations, strongly deviating from Pauling’s rule ofelectroneutrality. However, it conserves a reasonable BVS of 2.028(6)thanks to relatively large bond lengths with the coordinating cations(Table S1 in SI), making it the oxygenatom with the largest octahedral volume in the structure. Finally,it can be noted that Cu and Li occupy both 6 and 5-coordinate siteswith some cation mixing of Cu and Li in site 9 (36.7(2)% Li, see 2+ ions (Table S1 in SI).The energies of several phases with different Cu/Li orderings werestudied via density-functional theory (DFT) calculation, confirmingthat phases with Cu in sites 9 and 10 are indeed very close in energyof formation . The structure refinement also suggests about4.1(8)% and 9.4(6)% of oxygen vacancies in sites 5 and 6, respectively.This deviation from the stoichiometric composition and imperfect chemicalordering could point to inhomogeneities in composition and local structurein the sample, which would explain the asymmetric peak shapes associatedwith a distribution of cell parameters in the material.In the three structures, M and M′ sites are assembled inone plane 3a,d,g, cCu atoms 3a,b. Cu Cu atoms 3a. The eirection 3d. Turni–Sb–Sb)– 3g. Oxyge2CuO2, Li5SbO5, and Li4CuSbO5.5, one could expect to find a group/subgroup relationshipbetween the three structures. This is straightforward between Li2CuO2 and Li5SbO5, with thelatter belonging to a direct subgroup of the former through splittingof the Li, Cu, and O positions. The group–subgroup relationshipsof Li4CuSbO5.5 with Li5SbO5 and Li2CuO2 is implied by the correspondingBragg reflections that could match the structures of Li5SbO5 (C2/m) and Li2CuO2 (Immm), observed when preparingLi4CuSbO5.5 at 900 °C instead of 1100 °C(5SbO5 and Li2CuO2 phases (Table S2 in SI), but instead with those of Li4CuSbO5.5 prepared at 1100 °C. Here, the group–subgrouprelationship does not seem to be direct and its study would requireusing a large supercell of Li5SbO5 or Li2CuO2 given the more complex order found in Li4CuSbO5.5. Observing higher symmetry monoclinicand orthorhombic structures at lower temperature could point to animperfect ordering of cations in this sample. The difference in localstructure between the two samples prepared at 900 and 1100 °Cwas studied using two local structure probe techniques, namely X-raypair distribution function (XPDF) and extended X-ray absorption finestructure (EXAFS). Interestingly, the local atomic arrangements investigatedusing XPDF  show almost nodifference below a 20 Å radial distance for the two samples preparedat different temperatures. This suggests that Sb, the element whichscatters the strongest with X-rays, is ordered at intermediate rangesin the 900 °C sample. Clear deviations beyond 20 Å are consistentwith the different average long-range structures observed from theBragg data, which could correspond to the higher symmetry structuresof Li5SbO5 and Li2CuO2. The Cu K-edge EXAFS data  present largely similar local environments for Cu2+ cationswith an excellent overlap of the two sample’s signals up to R = 6 Å. These two local structural probes clearlyshow that the local chemical environments for both materials are essentiallyidentical, indicating that the square pyramidal configuration of Cuis strongly favored, as would be expected for this chemistry. Thedeviation at higher R in the EXAFS data would indicate that the long-rangeCu ordering is different in Li4CuSbO5.5 whenprepared at 900 and 1100 °C. Altogether, these data suggest thatthe ordering of cations and oxygen vacancies is incomplete at 900°C, leading to different long-range average structural models.Higher temperature helps to overcome kinetic barriers related to structuraldefects and to complete the long-range ordering of the structure.Giventhe similarity between the structures of Li 1100 °C1c. The r2.3I2O, CuIIO, NaCuIIIO2) and to the reported Li3CuSbO5 phase,11 which contains Cu2+ and Sb5+ in comparable coordination environments.The tendency for Cu to adopt strongly distorted coordination environmentsclearly appears on the Cu K-edge data, with intense pre-edge featuresthat overlap with the edge itself. Nevertheless, there is a good matchbetween the edge position of Li4CuSbO5.5 andCuO, and even more so with Li3CuSbO5 which showsa very similar edge shape as can be expected from the comparable environmentsin both materials. Li4CuSbO5.5 shows an additionalpre-edge feature at 8984 eV that can be interpreted as the presenceof Cu2+ in square pyramidal environment and is not observedin Li3CuSbO5 for which all Cu occupies distortedoctahedral environments. Measurements at the Sb K-edge (1-edge(inset), which can better resolve mixed oxidation states, unambiguouslyconfirm a +5 valence for antimony.Confirmationof Cu and Sb oxidation states was obtained through X-ray absorptionnear-edge spectroscopy (XANES) and magnetometry measurements. Measurementat the Cu K-edge 4a was cob K-edge 4b and Sb4CuSbO5.5 in an applied field of 100 Oe between 2 and 300 K shows a paramagneticbehavior which was fitted with a Curie–Weiss function modifiedwith a temperature-independent paramagnetic contribution between 50and 300 K. An effective magnetic moment of 1.313(3) μB per Cu atom was obtained, which is lower than the expected spin-onlyvalue for Cu2+ (μS.O. = 1.73 μB for S = 1/2). Isothermal field-dependentmagnetization curves measured between −70 and 70 kOe at 2,10, 50, and 300 K (2CuO2 impurity (<1 wt %) which can affectthe response of the material to the magnetic field. Even a small molefraction of ferromagnetic impurities can affect the measurement whileremaining undetectable by diffraction methods. To suppress the contributionof such impurities that saturate below 4 T, the magnetic susceptibilitydata measured at 60 kOe and 40 kOe were subtracted, and the data measuredbelow 50 K were discarded . Upon oxidation to 5 V vs Li+/Li, a charge capacityof 50 mAh/g is obtained with several features at 3.4, 4.3, and 4.9V  and a reversible discharge capacityof only 20 mAh/g with a cutoff at 1.5 V vs Li+/Li. Thetheoretical capacity for the extraction of one Li+ ioncorresponds to 90 mAh/g, suggesting that about 0.55 Li+ ions are extracted from the material and 0.22 are reinserted. Furthercycling in this voltage range improves only slightly the capacityup to 35 mAh/g reversible capacity after 15 cycles . The high-voltage plateau at 4.9 V vs Li+/Li is only observed on the first cycle and is reminiscent of theactivation plateau of Li-rich layered oxides corresponding to irreversibleoxygen oxidation. In addition to the presence of native oxygen vacanciesin the pristine structure, it is possible that additional oxygen isreleased upon high-voltage electrochemical oxidation instead of, orconcomitantly, with the oxidation of Cu2+ to Cu3+. In any case, this situation is not favorable for reversible insertionreactions.The propertiesof Li+/Li, a large capacity reaching 222 mAh/g is obtained with a longplateau at 1.2 V . The next chargeand discharge give a capacity of 237 and 226 mAh/g, respectively,with, however, a large voltage hysteresis (3.5 V) between dischargeand charge. From the second cycle, the low voltage plateau on reductionis now found at 1.75 V instead of 1.2 V vs Li+/Li. A reversiblecapacity between 220 and 270 mAh/g was maintained for 20 cycles beforeobserving a rapid capacity drop .Remarkably, when the material is discharged to 1 V vs Liin situ while first charging the material to 5 V, then discharging to 1V and charging again to 5 V .The capacities obtained for each step are lower than those measuredin coin cells , which can beexplained by the larger amount of material used and higher polarizationin the in situ cell, but they are representativeenough to observe the structural evolution of the material. Very littlechange is observed upon the initial charge to 5 V, which is not unexpectedgiven the small amount of Li (∼0.51) removed from the structure.Deinsertion of lithium, which is a weak X-ray scatterer, does notmake an observable difference to the intensity of Bragg peaks usinga lab X-ray source. However, we should be able to measure changesin lattice parameters or the creation of additional oxygen vacancies.This is not the case here. The following discharge to 1 V is markedby a strong reduction in peak intensity of all Bragg reflections correspondingto Li4CuSbO5.5. No new peaks were observed toreplace them; however, a general increase of the background contributioncan be noted, which suggests the formation of an amorphous phase ratherthan a new crystalline phase. This transition is incomplete, withsome peaks from the pristine phase still observed at the end of discharge.This can be understood from the lower capacity obtained in the insitu cell (171 mAh/g) compared to the coin cell (222 mAh/g) for whichthe lower polarization lets the reaction proceed to completion. Thesecond charge step shows only weak variations in the remaining peakintensity and positions as well as the background intensity. The amorphizationreaction is irreversible, and the structure of Li4CuSbO5.5 is not recovered upon oxidation. The large capacity obtainedis therefore likely to come from a conversion reaction that eventuallyleads to the cycling of an amorphous composite, thus explaining thelarge voltage hysteresis between discharge and charge.To shed further light on the structural evolution of thematerialupon (de)lithiation, XRD data were obtained 34CuSbO5.5 was preparedto explore the possibleincorporation of oxygen vacancies in a rocksalt oxide. Before discussingthis result, it is interesting to comment on the electrochemical propertiesof this material in light of other published work. First, we attributedthe large reversible capacity on reduction to a conversion reactionthat leads to an amorphous phase. It should be noted that the capacityof this reduction process (222 mAh/g) corresponds approximately tothe reaction of 2.5 Li+ ions with Li4CuSbO5.5. This is slightly more than the capacity expected for thefull reduction of Cu2+ to Cu0 (180 mAh/g), butconsistent with such a reaction. Several reports have mentioned thepossibility for Li+ to displace copper from a structureand form Cu nanoparticles.18 In a related example, Larcher et al. reported thatthe trirutile CuSb2O6 structure forms an amorphousLi2Sb2O6 phase and Cu nanoparticlesupon electrochemical reduction vs metallic Li.15 It is possible that Li4CuSbO5.5 followsa similar reaction pathway:6SbO5.5” does notcorrespond to a reported crystalline structure,and most likely forms Li5SbO5, Li7SbO6, and/or Li2O instead, although we cannotexclude an unknown composition for an amorphous phase. This reactionpathway could explain the short cycle life of the cell, as Li-richpentavalent Sb phases may form an electronically insulating matrixthat eventually prevents copper nanoparticles from reacting reversibly.Li+/Li may be explained by the Cu2+/Cu3+ redox couple or oxidation of oxygen. Multiple advanced characterizationtechniques would be required to fully pin down the charge compensationmechanism in this case, while maintaining a large degree of uncertaintygiven the small capacity associated with this process. Instead, wecan probe the possibility for oxygen evolution by evaluating the Gibbsenergy of 4CuSbO5.5, Li3CuSbO5,11 and Li2O:2O at 298 K (ΔG ≈ −610 kJ·mol–1)19 to obtain the energy of 4CuSbO5.5, oxygenrelease, and plating of one mole of lithium at the anode:3CuSbO5 is known to exist, and we pushed the cell voltage well beyond3.39 V. However, no signs of Li3CuSbO5 wereobserved by in situ XRD, which suggests that thisreaction may be kinetically limited. Indeed, this reaction requiresgood oxygen diffusion and a large reorganization of cation and anionlattices, moving from the rocksalt-derived structure of Li4CuSbO5.5 with oxygen vacancies (density of 4.5 g·cm–3) to the close-packed rocksalt structure of Li3CuSbO5 (density of 4.9 g·cm–3).Second, the capacity obtained upon oxidation between 4 and 5 Vvs Li4,20 which can be obtained by removinganother equivalent of Li2O from Li3CuSbO5 . Beyond their study of intercalationmaterials, these compounds could present interesting magnetic propertiescontrolled by the progressive dilution and different orderings ofthe S = 1/2 Cu2+ ions.It is interesting to note that another phase is compositionallyrelated to the two discussed above: the quantum-spin liquid LiCuSbOSbO5 see 7. This i2CuO2, Li2PdO2, LiCu2O2, Li3CuO3, Li3AuO3, LiCu3O3, Li3Cu2O4, Li5AuO4, Li5SbO5, Li5BiO5, Li6TeO6, Li6Zr2O7, and Li4Cu4O4 (Table S3 in SI).35 Li2NiO2, which is structurally related toLi2CuO2, could also enter this list but is moreoften described as a layered rocksalt containing excess Li in tetrahedralsites.36 The ordering of oxygen vacanciesin these structures directly depends on the proportion of unoccupiedsites in the oxygen sublattice and, most of the time, allows to maximizethe distance between vacancies. Examples of vacancy ordering patternsfor some of these structures are shown in Figure S7 in SI. Most structures can be described by a single hexagonalanion sublattice plane stacked with a given periodicity along the[111] direction of the corresponding cubic rocksalt cell. However,Li4CuSbO5.5 requires two different alternatingsublattice planes, one with a close-packed array of oxygen atoms andanother with only 5 out of 6 oxygen sites occupied. Among the structurescited above, only Li6Zr2O7 also requirestwo different sublattices planes.35 Thesmallest distance between two vacancies, normalized by the averagerocksalt a lattice parameter, is presented in 5SbO5 and Li5BiO5 correspondsto the a lattice parameter in the cubic parent. Li4CuSbO5.5, with 1 out of 12 oxygen sites in theunit cell being vacant, is the rocksalt structure with the smallestamount of ordered oxygen vacancy we were able to identify. It is noteworthythat Sr8Fe8O23, derived from theperovskite structure,37 maintains orderedoxygen vacancies with as little as 1 in 24 sites unoccupied. In thatcase, vacancies are separated by a distance of 2 × a, corresponding to the  vector in theequivalent perovskite lattice. Sr4Fe4O11 and Sr2Fe2O5, the n = 2 and 4 members of the oxygen-deficient SrnFenOn–13 series,37 follow a similar trend andcan be presented in a and vectors discussed above are also relevant in the cubic perovskitelattice. Given this comparison, it cannot be excluded that Li-richrocksalt oxides with more complex compositions could be prepared witha lower concentration of ordered oxygen vacancies than in Li4CuSbO5.5. In that regard, it should be noted that Greavesand Katib22 reported the preparation ofLi3.5Zn1.5SbO5.75□0.25 and Li3.5Zn1.5BiO5.75□0.25, that is, 1 in 24 oxygen sites unoccupied, with, however,oxygen vacancies statistically distributed over all oxygen positions.This result shows that (i) other divalent MII/pentavalentM′V cation couples may be used to prepare Li-richrocksalt oxides with oxygen vacancies and (ii) the amount of oxygenvacancies in Lix3+2MIIx2–2M′VOx6–□x may vary betweenLi3MII2M′VO6 (x = 0) and Li5M′VO5□1 (x = 1) that favor the formation of trivalentcations tend to give the well-known layered honeycomb Li5ReO6-type structure, as is the case for Cr, Al, and Gacompounds.39 However, by using reducingconditions to stabilize divalent cations, we can expect the formationof oxygen vacancies with other transition metals beyond Cu and Zn-based compounds for which normal atmospheric conditionsare sufficient. Two-step synthesis procedures, starting with the reactionof precursors at high temperature, followed by a second annealingstep under oxidizing or reducing conditions, have been successfulin the preparation of double perovskites with controlled oxygen vacancycontent .42 Transposing this approach to the preparation of Li-rich rocksaltoxides could lead to a better understanding of defect formation inthis structural family. Reciprocally, the discovery of Li-rich rocksaltoxides Li4MM′Ox6– can be used to prepare functional double perovskite A2MM′Ox6– by simple metathesis solid-state routes using ACl2 salts.44 This approach enables the formation of double perovskite with newcation ordering patterns at reduced temperatures (700–900 °C)compared to conventional synthesis routes (>1000 °C).Focusing now on the existence of Li-rich rocksalt oxides with orderedoxygen vacancies, several materials have been reported in the literature,although not always identified as rocksalt-derived structures. Identifyingsuch structures from an electronic database remains challenging giventhat we are searching for a “missing” structural feature; therefore, some structures may have been unintentionallyexcluded from our search. Examples of materials with only one cationin addition to lithium and ordered oxygen vacancies are Li1/6□1/6Mn2/3)O245 and phasesderived from the Li4FeSbO6 composition.46 These examples pointed out that cation vacanciesin a rocksalt structure are found when an overstoichiometry of anionscompared to metal atoms is required to ensure electroneutrality. Thisis more likely to be achieved in relatively oxidizing conditions,in opposition with the formation of structures with oxygen vacancies.Formation of oxygen vacanciesis not the only mechanism to balanceionic charges in Li-based rocksalt structures. Another known mechanismis the formation of cation vacancies as studied for materials such as Li, CuO , and Sb2O3 . All precursors are kept in a drying oven at 200 °Cbefore use. The precursors in the proportions Li:Cu:Sb = 4.4:1:1 areeither hand ground or ball-milled using a planetary mill with thesame outcome. They are then fired in air at 1100 °C for 12 h.The excess of Li2CO3 used plays a role in stabilizingLi4CuSbO5.5 as longer heating at 1100 °Cwill result in Li2O loss from the compound to form Li3CuSbO5 according to the reaction Li4CuSbO5.5 → Li3CuSbO5 + 1/2Li2O. Rapidly quenching the sample from 1100 °C betweenstainless steel plates or slowly cooling (0.1 °C/min) it from850 °C to room temperature leads to small variations in cellparameters of the sample (see Table S2 inSI). The low-temperature sample was prepared with the same procedure,using a firing temperature of 900 °C instead of 1100 °C.For structural characterization by synchrotron X-ray and neutron diffraction,a sample was prepared using enriched 7Li2CO3  to reduce the absorptionof neutrons due to 6Li in the sample. The material wasmanipulated in air but kept in an Ar-filled glovebox during storageto prevent building a Li2CO3 layer at the surfacewith prolonged contact with ambient air.Li5.21, λ = 1.78901Å) in Bragg–Brentano geometry with sample rotation. SXRDwas performed at the I11 beamline at Diamond Light Source , with an incident wavelength of 0.82468(1) Å using five multianalyzercrystal detectors. The samples were sealed in Ø = 0.3 mm glasscapillaries and spun during measurement. Time-of-flight (ToF) neutronpowder diffraction data were collected on the HRPD instrument at ISISneutron source . Samples were sealed in Ø =6 mm vanadium cylindrical cans in an argon-filled glovebox. Indexingwas performed using the DICVOL method as implemented in the Fullprofsuite.14 A simulated annealing method wasused to obtain initial atomic positions with the FOX program.13 The structural model was completed and refinedby the Rietveld method48 and using Fourier differencemaps as implemented in the Fullprof suite.14 In situ XRD was performed using an electrochemical cell equippedwith a Be window (250 μm thick) and an Al current collector(3 μm thick) on a Rigaku SmartLab diffractometer with a 9 kWrotating anode providing a parallel beam of Mo Kα1 radiation (λKα1 = 0.709032 Å). X-raytotal scattering measurements were done at the I15-1 (XPDF) beamlineat Diamond Light Source . Samples were loaded intoquartz capillaries with a 1 mm inside diameter and measured whilespinning. Data were collected using a PerkinElmer XRD1611 CP3 areadetector with an active area of 409.6 × 409.6 mm2 witha Q range of 36 Å–1. Pairdistribution functions were calculated using GudrunX using the appropriatecomposition, background data, instrument corrections, and a Q max of 30 Å–1.Routine analysis of phasepurity and lattice parameters were performed on a Panalytical diffractometerwith a monochromatic Co source (Kα5.34CuSbO5.5), conductive carbon , and binder  in proportions85:10:5 in weight. For ex situ characterization by XRD, the activematerial was simply mixed with 10 wt % C65 conductive carbon and usedas a powder. Active material loadings were typically between 5 and10 mg. Metallic Li was used as an anode, LP30 (1 M LiPF6 in EC:DMC 1:1) was used for the electrolyte, and Whatman GF/D borosilicateglass fiber membranes dried under vacuum at 300 °C for 24 h asthe separator. All parts were assembled in an Ar-filled glovebox.Galvanostatic cycling was performed at a C/20 rate  between 1 and 5 V vs Li+/Li.After cycling, samples for ex situ characterization were recoveredinside the glovebox, washed three times in anhydrous DMC, and driedunder vacuum.Electrochemical characterizationwas performed in two-electrode Swagelokcells and 2025-type coin cells. The positive electrode consisted ofa laminated mixture of active material (Li5.449 Fourier transformof the EXAFS Cu K-edge data was done using a sine window functionfrom 3.8 to 12.7 Å–1 (k-range).Pelletsof sample diluted with cellulose were prepared with an optimized densityfor X-ray absorption measurements at the Cu K-edge, Sb K-edge, andSb L1-edge. X-ray absorption spectra were measured at the B18 beamlineat Diamond Light Source  in transmission mode. Thespectra were calibrated by fixing at 8979/30491/4966 eV, the maximumof the derivative of Cu/Sb/Ti metal foil references placed after thesamples for the corresponding edges, respectively, and normalizedwith the Athena software.5.5M(H) loops were measured between −70 and 70 kOe at 2, 10, 50,and 300 K.About 30–40mg of freshly prepared sample in the form of a powder was sealed ina high purity quartz tube .Magnetic measurements were carried out using a commercial superconductingquantum interference device magnetometer MPMS3 .The contribution of the quartz tube to the magnetization was confirmedto be negligible prior to measuring the sample. Zero-field cooledand field cooled measurements were first performed at 100 Oe from2 to 300 K, followed by field-cooled measurements at 40 kOe and 60kOe. Finally, magnetic field-dependent magnetization 5.650 with PAW pseudopotentials.51 The atomic and vacancy positions in the structures with mixed siteoccupancies, Li4CuSbO5.5 and Li3CuSbO5 were determined by, first, optimizing the geometries of possibleatomic configurations in 2 × 2 × 1 supercell structures, the interatomicforces are reduced below 10–3 eV/Å, and thencomparing their total energies. The lowest energies were used in k-points sampling,and LDSA53 approach to account for stronglycorrelated 3d electrons in Cu, with Hubbard U = 9.79eV and J = 2.5.54All calculationswere conducted with DFT as implemented in VASP-5.4.4


The crystal structure and grain size of Co40Fe40W10B10 films with different thicknesses and annealing temperatures are observed and estimated by an X-ray diffractometer pattern (XRD) and full-width at half maximum (FWHM). The XRD of annealing Co40Fe40W10B10 films at 200 °C exhibited an amorphous status due to insufficient heating drive force. Moreover, the thicknesses and annealing temperatures of body-centered cubic (BCC) CoFe (110) peaks were detected when annealing at 250 °C with thicknesses ranging from 80 nm to 100 nm, annealing at 300 °C with thicknesses ranging from 50 nm to 100 nm, and annealing at 350 °C with thicknesses ranging from 10 nm to 100 nm. The FWHM of CoFe (110) decreased and the grain size increased when the thickness and annealing temperature increased. The CoFe (110) peak revealed magnetocrystalline anisotropy, which was related to strong low-frequency alternative-current magnetic susceptibility (χac) and induced an increasing trend in saturation magnetization (Ms) as the thickness and annealing temperature increased. The contact angles of all Co40Fe40W10B10 films were less than 90°, indicating the hydrophilic nature of Co40Fe40W10B10 films. Furthermore, the surface energy of Co40Fe40W10B10 presented an increased trend as the thickness and annealing temperature increased. According to the results, the optimal conditions are a thickness of 100 nm and an annealing temperature of 350 °C, owing to high χac, large Ms, and strong adhesion; this indicates that annealing Co40Fe40W10B10 at 350 °C and with a thickness of 100 nm exhibits good thermal stability and can become a free or pinned layer in a magnetic tunneling junction (MTJ) application.This research explores the behavior of Co C) [C) and high saturation magnetization (MS) was obtained [40Fe40W10B10 films deposited on Si (100) substrates when an annealing process occurs at 200 °C, 250 °C, 300 °C, and 350 °C, respectively. This experiment elected to add B and W into CoFe material and investigated their specific properties, including structure, adhesion, and magnetic characteristics, after annealing treatments.Ever since the conception of nanocrystalline materials by Rozlin in 2012, the CoFe alloy found in magnetic materials has presented excellent soft magnetic properties, categorized by high saturation magnetization (Ms) and a high Curie temperature (TC) ,4,5,6. TC) ,10,11,12C) . An expeC) . The PMAobtained . Findingobtained . Howeverobtained . Higher 40Fe40W10B10 with a thickness of 10–100 nm was sputtered onto Si(100) substrate at room temperature (RT) by a magnetron DC sputtering direct method of 50 W power and under the following four conditions: (a) annealed at a treatment temperature (TA) of 200 °C for 1 h, (b) annealed at 250 °C for 1 h, (c) annealed at 300 °C for 1 h, and (d) annealed at 350 °C for 1 h. The chamber base pressure was 8.5 × 10−7 Torr, and the Ar working pressure was 3 × 10−3 Torr. The pressure in the ex-situ annealed condition was 3 × 10−3 Torr with a selected Ar gas. The target alloy composition of CoFeWB was Co (40%), Fe (40%), W (10%), and B (10%). The structure of the CoFeWB thin films was detected by grazing incidence X-ray diffraction (XRD) patterns obtained with CuKα1  and a low angle diffraction incidence of roughly 2 degrees. The in-plane low-frequency alternate-current magnetic susceptibility (χac) and hysteresis loop of the Co40Fe40W20 were studied using an χac analyzer  and an alternating gradient magnetometer . Moreover, in χac measurement, the χac analyzer was used to calibrate the standard sample under the action of an external magnetic field. Then, the sample was inserted into the χac analyzer. The driving frequency was between 10 and 25,000 Hz. χac was measured using magnetization. All test samples had an equivalent shape and size to eliminate demagnetization. The χac valve acted as an arbitrary unit (a.u.) because the AC result corresponded to the reference standard sample and could have been a comparison value. The connection between magnetic susceptibility and frequency was measured by an χac analyzer. The best resonance frequency (fres) was measured by an χac analyzer and represented the frequency of the maximum χac. Before measurement, the contact angle was properly air cleaned on the surface. The contact angle of the CoFeWB film was measured with deionized (DI) water and glycerol. The contact angle was measured when the samples were taken out of the chamber. The surface energy was obtained by measuring the contact angle and employing specific calculations [Coulations ,19,20.40Fe40W10B10 thin films when analyzed for the crystal structure by XRD at the annealed temperatures of 200 °C, 250 °C, 300 °C, 350 °C, and 400 °C, respectively. The Scherrer formula isIn the formula, k (0.89) is Scherrer’s constant; λ is the X-ray wavelength of the Cu Kα1 line; B is the FWHM diffraction CoFe (110) peak; and θ is the half-angle of the diffraction peak. 40Fe40W10B10 thin films under four annealed conditions, with thicknesses ranging from 10 to 100 nm. The external magnetic field of 200 Oe in the plane is enough to observe the saturation magnetic spin state. The figure shows low coercivity (HC), which indicates that Co40Fe40W10B10 films are soft magnetic. The saturation magnetization (MS) of Co40Fe40W10B10 thin films under four post-annealing conditions illustrates the magnetic properties of Co40Fe40W10B10 thin films that were measured by AGM, as shown in 40Fe40W10B10 films, Ms increases with the increase of thickness, indicating the effect of thickness on Ms. It was also found that the Ms of the CoFeWB thin films increased by raising the annealing temperature. Apparently, the MS, when annealed at 350 °C, is much larger than other conditions because of magnetocrystalline anisotropy [S value of Co40Fe40W10B10 thin films is increased to 350 °C, which shows that the thermal stability of Co40Fe40W10B10 thin films is better than that of other research findings [isotropy ,27. CoFeisotropy ,29. In tfindings .ac) result of CoFeWB films with thicknesses ranging from 10 to 100 nm under four conditions. The low frequencies were measured in the range of 50–25,000 Hz. The results display that the χac values decrease with an increasing frequency (Hz) under the four conditions. The corresponding maximum χac values of various CoFeWB thicknesses under four conditions are shown in ac value was 0.24 when the thickness was 100 nm at 200 °C; the maximum χac value was 0.26 when the thickness was 100 nm at 250 °C; the maximum χac value was 0.36 when the thickness was 100 nm at 300 °C; and the maximum χac value was 1.17 when the thickness was 100 nm at 350 °C. These results clearly display an increased χac owing to the thickness effect and magnetocrystalline anisotropy in the CoFeWB films. res) of CoFeWB. The maximum χac demonstrates that the spin sensitivity is highest at the optimal resonant frequency [ac peak indicates the spin exchange-coupling interaction and dipole moment of the domain under frequency. Additionally, the ƒres value is below 1000 Hz, which makes CoFeWB films for applications in soft magnetism devices. It was found that the ƒres values of all CoFeWB thicknesses were in the range of 50–1000 Hz, suggesting the maximum χac had the strongest spin sensitivity at this frequency [requency . The χacrequency .40Fe40W10B10 thin films were less than 90°, representing that Co40Fe40W10B10 film exhibits good hydrophilicity and wettability. The surface energy of thin films is an important parameter because it relates to the adhesion of thin films. When CoFeWB thin film is used as a seed, buffer, free, or pinned layer, the strong adhesion of the thin films is essential. The data of contact angles are used to calculate the surface energy via Young’s equation [The contact angles were measured using DI water and glycerol. The data are presented in equation ,20:σsg =Here, σsg is the surface free energy of the solid; σsl denotes the liquid–solid interface tension; σlg is the surface tension of the liquid, and θ is the contact angle.40Fe40W10B10 thin films. These data of surface energy are shown in 2 at 50 nm, which was the highest value. When the post-annealing temperature was 250 °C, the highest surface energy of 60 nm was 31.01 mJ/mm2. When the post-annealing temperature achieved 300 °C, it was 50.56 mJ/mm2 at 100 nm. When the post-annealing temperature achieved 350 °C, it was 54.95 mJ/mm2 at 100 nm. When the surface energy is high, the liquid absorption capacity of the surface is correspondingly high. High surface energy corresponds to strong adhesion [40Fe40W10B10 properties and compare them with other specific Co40Fe40V10B10 film magnetic properties and surface energies, as mentioned in ac and surface energy of 100 nm Co40Fe40W10B10, annealed at 350 °C, is larger than that of the Co40Fe40V10B10 film, indicating that Co40Fe40W10B10 thin film is more suitable as a free or pinned layer in MTJ and can be applied in MRAM applications.adhesion . Surfacec and in-plane magnetization because thicker CoFeWB thicknesses showed a large in-plane demagnetization field. Ms and χac also increased as the thickness and annealed temperature increased. The χac and MS of films annealed at 350 °C are much larger than in other conditions, owing to the thickness effect and magnetocrystalline anisotropy in the CoFeWB films. Alloying additions of W improved the thermal stability of CoFeB films. The maximum Ms and χac values were achieved for CoFeWB films with a thickness of 100 nm after annealing at 350 °C. The fres values of all films were less than 1000 Hz. The contact angles of CoFeWB were smaller than 90°, indicating that the films were hydrophilic and had a good wetting effect. The surface energy of a high post-annealing temperature was larger than that of a low annealing temperature. As the post-annealing temperature increased, the surface energy clearly increased. Based on the magnetic properties and surface energy, the optimal condition of CoFeWB film is a thickness of 100 nm with an annealed temperature of 350 °C, which is suitable as a free or pinned layer in MTJ for magnetic component applications.XRD patterns revealed that CoFeWB thin films are composed of an amorphous status when annealed at a temperature of 200 °C. BCC CoFe (110) peaks were detected when annealing at 250 °C with thicknesses ranging from 80 nm to 100 nm, annealing at 300 °C with thicknesses ranging from 50 nm to 100 nm, and annealing at 350 °C with thicknesses ranging from 10 nm to 100 nm. The intensity and grain size of CoFe (110) peaks generally increased with increasing film thickness and annealing temperatures, indicating a development in crystallographic texture. CoFeWB films presented soft magnetism owing to low H


This study was performed on 18 FeLV-infected cats with mediastinal lymphoma. The complete blood count, creatinine, alanine aminotransferase, and CD4+/CD8+ ratio were measured four times before treating with chemotherapy in the 1st, 2nd, 3rd, and 4th weeks. The white blood cell (WBC) counts, neutrophils, packed cell volume (PCV), and mean corpuscular volume (MCV) changed, while the CD4+/CD8+ ratio was not found to be significantly different. These results suggested that COP chemotherapy is safe as a treatment for FeLV-infected cats with mediastinal lymphoma.Mediastinal lymphoma is the most commonly diagnosed tumor in cats with feline leukemia virus infection (FeLV). The cyclophosphamide, vincristine, and prednisolone (COP) chemotherapeutic protocol is widely used and has achieved high complete-response (CR) rates with a long disease-free interval (DFI), and a long median survival time (MST), with low toxicity. The CD4+/CD8+ ratio, and the mortality of 18 client-owned FeLV-infected cats with mediastinal lymphoma. The complete blood count, creatinine, alanine aminotransferase, and CD4+/CD8+ ratio were measured four times before treating with chemotherapy in the 1st, 2nd, 3rd, and 4th weeks. The white blood cell (WBC) counts at the 1st week were significantly different from the 2nd, 3rd, and 4th inductions . The neutrophils at the 1st week were significantly different from the 2nd and 3rd inductions . The packed cell volume (PCV) at the 1st week was significantly differed from the 2nd, 3rd, and 4th induction times  at the 1st week was significantly different from the 4th week (p = 0.0145). We found that chemotherapy did not cause any significant change in the CD4+/CD8+ ratio . The Kaplan–Meier curves showed the median survival time (MST) for the cats with a CD4+/CD8 ratio of less than 1 after the 1st week of chemotherapy was 134 days. This suggested that COP chemotherapy was a safe treatment for FeLV-infected cats with mediastinal lymphoma.The objective of this study was to evaluate the effect of the COP chemotherapeutic protocol on hematological parameters, CD4 Lymphoma, one of the most common tumors in cats ,2,3,4, i+ and CD8+ T cells are the main immune cell lymphocytes (cell-mediated immunity [CMI]). They play a major role in the tumor-response process [+/CD8+ ratio in cancer patients is considered one of the T-cells immune risk phenotypes which is related to an increase in morbidity and mortality rate in humans [+/CD8+ ratio is used to assess immunity, including the pregression of feline immunodeficiency virus (FIV) and FeLV. FIV-infected cats have an inverted CD4+/CD8+ ratio that is less than 1 due to the ratio involving a selective reduction in CD4+ T cells, while CD8+ cells are relatively normal [+/CD8+ ratio determined from flow-cytometric analysis from 39 normal cats was 1.13. Unlike FIV, FeLV did not cause an inversion of the CD4+/CD8+ ratio [+/CD8+ ratio in bone marrow and blood was 1 [+/CD8+ ratios in bone marrow-positive and -negative follicular lymphoma patients were 1.53 and 1.57, respectively [Depleted T cell populations have been reported during cancer progression in humans . CD4+ an process ,14,15. An humans . In catsy normal . The med8+ ratio ,18. A stod was 1  and 1.5 od was 1 , respectectively . From ou+/CD8+ ratio, and mortality in FeLV positive cats with mediastinal lymphoma.The objectives of this study were to evaluate the effect of the COP chemotherapeutic protocol on hematological parameters, the CD4This research was approved by Kasetsart University’s Institutional Animal Use and Care (ACKU 61-VET-090). Informed consent from clients was obtained in all cases. Eighteen client-owned cats with mediastinal lymphoma were enrolled in the study. Mediastinal lymphoma was confirmed using the combination test of thoracic radiography or ultrasonography and the cytological analysis of pleural effusion during May 2017 and May 2019 at the Kasetsart University Veterinary Teaching Hospital, Bangkok, Thailand. The staging was based on liver or spleen involvement after performing ultrasonography . The asp2, IV) cyclophosphamide , and prednisolone . Additionally, all cats in this study received recombinant feline interferon omega (rFeIFN-ω) at an oral dose of 10,000 unit/day, during chemotherapy. Clinical signs were observed to obtain the clinical score (CS) at the 1st week and 4th week. CS were classified to 3 clinical groups (CG): CG1, with CS = 0 (asymptomatic); CG2, with CS = 1–5 (mild disease); CG3, with CS ≥ 6 (severe disease) [® FeLV-FIV) were performed, and relevant data were recorded for all cats by licensed veterinarians. Cats that were negative using the test kit FeLV antigen had a whole-blood PCR analysis for FeLV provirus. Approximately 3 mL of whole blood was collected from the jugular vein, cephalic vein, or median saphenous vein and distributed to ethylenediaminetetraacetic acid (EDTA)-coated tubes and plain glass tubes for the analysis of complete blood count (CBC) and blood-smear examination, serum biochemistry panel , and measurement of CD4+ T-cells and CD8+ T-cells before performing chemotherapy in the 1st, 2nd, 3rd, and 4th weeks of the treatment. CBC was performed using an automated hematology analyzer . Serum biochemistry was performed using a laboratory chemical analyzer .All enrolled cats received COP chemotherapeutic protocol as previously described . All catdisease) . CompletIn the 4th week, before the maintenance phase, treatment responses were evaluated using chest radiography or ultrasonography and clinical signs. Cats were categorized as having a complete response , partial response , or no response  . After t®, Corning, NY, USA) and mixed with monoclonal antibodies against feline CD4+  and CD8+ . After vortexing, the mixture was incubated in a dark room for 15 min. Then, 1000 µL of 1% lysis buffer  was added to lyse the red blood cells. The mixture was centrifuged at 4 °C and 1500 rpm for 5 min. The pellet was washed with 3000 µL of phosphate-buffered saline pH 7.4  and centrifuged at 4 °C and 1500 rpm for 5 min. The cell pellet was reconstituted and fixed with 200 µL of 1% paraformaldehyde (Sigma-Aldrich) and stored in a dark room until analysis using the flow cytometer . The analysis of the lymphocyte subset (CD4+ and CD8+) used the flow cytometer at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand. The ratio was considered to be decreased when it was <1.Each whole blood sample (3 mL) was immediately mixed with EDTA to prevent blood clotting. After this, 50 µL of the blood mixture was transferred to a 5 mL round-bottomed polystyrene tube  and was also used to evaluate the clinicopathological findings after four weeks for the cats with mediastinal lymphoma. Fisher’s exact test was used to determine the association between the CD4+/CD8+ ratio in the 1st week and mortality before receiving chemotherapy in cats with mediastinal lymphoma and was also used to determine the association between the CD4+/CD8+ ratio at the 4th week and clinical response to chemotherapy. MST was estimated based on Kaplan–Meier curves. Overall survival (OS) was determined from the first dose of chemotherapy to death related to mediastinal lymphoma. Unrelated deaths were censored, along with those lost to follow-up. p-values less than 0.05 were considered statistically significant.Commercially available statistic software packages  were used for statistical analyses. A normality test was performed for all data using the Shapiro–Wilk W test. The Friedman test was used to evaluate the effect of chemotherapy on the CD4® 10,000-unit, PO).All 18 enrolled cats were positive for the FeLV antigen. Patient characteristics are shown in 3 cell/µL (range 8–29 × 103 cell/µL), 8.55 × 103 cell/µL (range 3.67–19.2 × 103 cell/µL), 6.55 × 103 cell/µL (range 3.73–20.8 × 103 cell/µL), and 8.79 × 103 cell/µL (range 3.08–17.8 × 103 cell/µL), respectively. The results showed that there was a significant difference among the four times for WBC .After the CBC measurement , values 3 cell/µL (range 1.29–23.2 × 103 cell/µL), 6.53 × 103 cell/µL (range 1.47–14.59 × 103 cell/µL), 4.3 × 103 cell/µL (range 2.36–16.85 × 103 cell/µL), and 6.65 × 103 cell/µL (range 1.85–15.66 × 103 cell/µL), respectively. There was a significant difference among the 4 times for the neutrophil count .The median neutrophil counts in the 1st, 2nd, 3rd, and 4th weeks were 12.07 × 103 cell/µL (range 1.02–9.41 × 103 cell/µL), 1.88 × 103 cell/µL (range 0.04–6.27 × 103 cell/µL), 1.57 × 103 cell/µL (range 0.59–4.31 × 103 cell/µL), and 1.21 × 103 cell/µL (range 0.11–5.02 × 103 cell/µL), respectively. There was no significant difference for the lymphocyte count between the four weeks .The median packed-cell volumes (PCVs) in the 1st, 2nd, 3rd, and 4th inductions were 38.5% (range 26.9–50.1%), 32.65% (range 22.1–41.1%), 31.7% (range 20.3–39.7%), and 32.45% (range 21.1–37.7%), respectively. There was a significant difference among the four times of measurement for the PCV (p = 0.0078) (p = 0.0145).The median mean corpuscular volumes (MCVs) in the 1st, 2nd, 3rd, and 4th weeks were 50.11 fL (range 42.2–57.45 fL), 50.17 fL (range 41.99–57.11 fL), 51.87 fL (range 43.31–56.18 fL), and 51.58 fL (range 43.29–58.92 fL), respectively. There was a significant difference in MCV ( 0.0078) B, and thp = 0.8121; The median mean corpuscular hemoglobin concentrations (MCHCs) in the 1st, 2nd, 3rd, and 4th weeks were 32.28 pg (range 28.77–35.5 pg), 31.95 pg (range 30.17–33.87 pg), 31.74 pg (range 29.17–34.28 pg), and 32.15 pg (range 28.34–34.77 pg), respectively. However, there was no significant difference in MCHC , 1 (range 0.32–4.17), 1.07 (range 0.45–2.57), and 1 (range 0.34–5.5), respectively. There was no significant difference among the 4 times for the CD4+/CD8+ . At the end of the study, 5 (27.78%) out of the 18 cats had died and 13 (72.22%) cats were still alive. There was no significant association between the CD4+/CD8+ ratio at the 1st induction and mortality (p = 1.000).The treatment response was evaluated before the 4th induction time point using thoracic radiography or ultrasonography, with 15 (83.33%) cats having a complete response and three (16.67%) cats having no response. However, there was no significant association between the CD4+/CD8+ ratio after the 1st week of chemotherapy of less than 1 (n = 7). There was one dead cat for cats with a CD4+/CD8+ ratio after the 1st week of chemotherapy that was greater than or equal to 1 (n = 11). The Kaplan–Meier curve showed that the MST for the cats with a CD4+/CD8+ ratio after the 1st week of chemotherapy of less than 1 was 134 days (p = 0.0173).There were five dead cats and 13 living asymptomatic cats after treatment for 52 weeks. There were four dead cats for cats with a CD4134 days . The Cox+/CD8+ ratio in 18 cats with progressive FeLV infection. Feline mediastinal lymphoma occurs most frequently in young male cats with FeLV infection [Despite the large number of feline lymphoma studies, there is a paucity of information regarding cats with a high prevalence of FeLV infection . In thisnfection ,23,26,27nfection . Howevernfection ,11. As iThe COP chemotherapeutic protocol, which is popular and commonly used to treat lymphoma in cats ,2,8, has+/CD8+ ratio) in FeLV-infected cats is scarce. The evaluation of toxicity based on CHOP  [p = 0.0179, and p ≤ 0.0001, respectively). This may have been due to the adverse effect of chemotherapy. The chemotherapeutic agents associated with neutropenia in the present study may have been resultant of the vincristine and cyclophosphamide. Vincristine, a cell cycle-specific drug, can interfere with microtubule formation and mitosis, causing the lowest neutrophil counts within 7–9 days [Chemotherapy is usually well tolerated by cats and is mainly based on two phases, namely, induction and maintenance. However, the toxicity information associated with COP chemotherapy during the induction phase, based on the CBC, blood chemistry and immunological parameters (CD4isolone)  and LOPH7–9 days . On the 7–9 days . The PCV7–9 days  from ski7–9 days .+/CD8+ at all 4 times during the induction phase with COP chemotherapy were not significantly different. In this research, FeIFN-ω may have played a role in modulating the host’s immune system, resulting in an unchanged ratio of CD4+/CD8+. According to another study, FeIFN-ω can improve clinical signs and hematologic parameters in naturally retroviral infected cats, but it did not change hypergammaglobulinemia, the pro-viral load and anemia, suggesting an overall effect on the innate immune reaction, rather than on acquired immunity [+/CD8+ ratio) in another study [+/CD8+ ratio of renal cell carcinoma patients treated with vinblastine in combination with human IFN-α was higher compared to those that had been treated with vinblastine alone [The ratios of CD4immunity . T-helpeimmunity . FeLV orer study , similarer study  and surver study . The CD4ne alone . However+/CD8+ ratio, tolerability, and survival time. This study was limited by the consideration of only one breed of cat, namely, the domestic short hair, and a relatively small number of specimens (n = 18). There was also a limit to the duration of the study. The availability of information on the CD4+/CD8+ ratio in cats with mediastinal lymphoma that have been treated using chemotherapy is inadequate due to limited studies. Therefore, further studies should be conducted to provide more data that may help to direct further treatment. Another limitation is that the staging of bone marrow, spleen, or liver involvement has not been performed. This is because all of the enrolled cats had suffered dyspnea due to mediastinum mass compression. The diagnosis and treatment must therefore be performed immediately. In order to avoid the bone marrow, spleen, or liver involvement, we enrolled the cats with single node (mediastinum) involvement.We observed that the COP chemotherapy in this study was well tolerated by the cats during the induction phase. More studies should be conducted with a longer observation period to evaluate the relationships among the CD4+/CD8+ ratio during the induction phase of COP chemotherapy in cats with mediastinal lymphoma was not different from pretreatment, though it can cause changes in the WBC, neutrophils, PCV, and MCV. These results suggested that COP chemotherapy is safe to apply for FeLV-infected cats with mediastinal lymphoma.The CD4


Staphylococcal Protein A (SpA), Streptococcal Protein G (SpG) and Finegoldia Protein L (PpL) are thought to only bind specific regions of human antibodies, allowing for selective purification of antibody isotypes and chains. Clinically, these superantigens are often classified as toxins and increase the virulence of the producing pathogen through unspecific interactions with immune proteins. To perform an in-depth interaction study of these three superantigens with antibodies, bio-layer interferometry (BLI) measurements of their interactions with a permutation panel of 63 IgG1 variants of Pertuzumab and Trastuzumab CDRs grafted to the six human Vκ and seven human VH region families were tested. Through this holistic and systemic analysis of IgG1 variants with various antibody regions modified, comparisons revealed novel PpL–antibody interactions influenced by other non-canonical antibody known light-chain framework regions, whereas SpA and SpG showed relatively consistent interactions. These findings have implications on PpL-based affinity antibody purification and design that can guide the engineering and understanding of PpL-based microbiota-immune effects.Immunoglobulin superantigens play an important role in affinity purification of antibodies and the microbiota-immune axis at mucosal areas. Based on current understanding,  Streptococcal bacteria [Staphylococcus aureus, which binds to the heavy chain constant region of IgG1, 2, and 4 and the variable heavy (VH) 3 framework (VH3) [Finegoldia magna (previously known as Peptostreptococcus magnus), which binds to the variable light kappa κ (Vκ) chain families 1,3,4 [B cell superantigens bind antibodies to hyperstimulate populations of B cells independent of T cells and have been widely used for antibody affinity purification . Superanbacteria , which brk (VH3) ,6; and Pes 1,3,4  at the fes 1,3,4 . When boes 1,3,4 , possibles 1,3,4 . They caes 1,3,4 ,12).®), a humanised monoclonal IgG1 antibody with its V-region derived from rodents [®) [With both IgG and Vκ being the predominant isotypes in humans ,14, supe rodents , is ofteents [®)  in clinients [®)  and haveGiven the common use of these three superantigens, a holistic ,20 and sE. coli (DH5α) strains [All Trastuzumab and Pertuzumab VH and Vκ CDR-grafted sequences used were described previously ,22. Brie strains  followed strains  , 30 s); Initial Baseline ; Baseline ; Association ; Dissociation ; Regeneration , 30 s) as previously described [Measurements of the rates of association (ka) and rates of dissociation (kd) were performed using PpL , SpA , and SpG  biosensors to Pertuzumab and Trastuzumab IgG1 variants described above in solution using the Octet Red96escribed ,23,24,27To examine the potential holistic effects of Vκ1-6 pairing with VH1-7 on antibody interactions with SpA, recombinant Pertuzumab and Trastuzumab IgG1 variants of the various pairings were studied. Notably, SpA is known to bind to the CH2 and CH3 domains of the heavy chain constant (CH).−9 M and 0.24–2.30 × 10−9 M, respectively , followed by Vκ3 (5.55–38.03 × 10−9 M) and Vκ4 (13.09–74.56 × 10−9 M) (refer to −9 M) followed by Vκ3 (3.67–17.77 × 10−9 M) and 4 (7.22–14.18 × 10−9 M). Note that Trastuzumab IgG1s showed lower and a narrower KD range than the Pertuzumab Vκ-VH equivalents suggesting effects from the CDRs which were what differed between the two sets of IgG1s.As a control for expected superantigen interactions, the Pertuzumab IgG1s of Vκ1, 3, and 4 interacted with PpL, where Vκ1 showed the lowest KD range (0.53–0.76 × 10−9 M), 6 (15.79–43.46 × 10−9 M) and certain Vκ2 variants  had KDs comparable to Vκ3 and 4 variants (5.55–74.56 × 10−9 M). Pertuzumab Vκ2 paired with VH5 and 6  had KDs comparable to Vκ1 (0.53–0.76 × 10−9 M) while Vκ2 paired with VH1 had the highest KD (poorest binding) of 148.67 × 10−9 M. There were also non-binding IgG1s of the Pertuzumab Vκ6 variant with VH1–3 (Poor Response) despite measurable responses when paired with VH4, 5 and 7.Unexpectedly, certain Pertuzumab VHs paired with Vκ2, 5 and 6 exhibited interactions with PpL. Amongst these Vκ families, Pertuzumab variants Vκ5 (13.58 & 13.88 × 10−9 M) and 6 (14.84–82.16 × 10−9 M) and Vκ2 paired with VH2 & 7  had KD values comparable to Vκ3 and 4 (3.67–14.18 × 10−9 M). Trastuzumab Vκ2 paired with VH1 and 6 had the highest KD (poorest interaction) at 124.41 & 190.4 × 10−9 M, respectively. The non-binders were Trastuzumab Vκ2 paired with VH3–5 (Poor Response) rather than in the Vκ6 family observed for Pertuzumab. These differences demonstrated a role of the CDRs and their contribution to the PpL engagement.Generally, the Pertuzumab trends were largely similar to the Trastuzumab IgG1s where KDs of Vκ5 (1.75 × 10We set out to investigate the interactions of antibody superantigens Protein A, G, and L systematically and holistically with the engineering of the various V-regions of IgG1 antibodies. By using CDRs of Pertuzumab and Trastuzumab grafted onto Vκ1-6 and VH1-7 FWRs, measurements to the superantigens showed no major differences for SpA between Pertuzumab and Trastuzumab IgG1 variants . This wa® measurements. Nonetheless, future studies can utilise other immunoaffinity resins or the Melon gel if they wish to confirm these findings. Notably, our size exclusion purification of monomeric IgG fractions does not show residual binding of SpG to interfere with our subsequent measurements and that affinity purifications do not rely on the small changes in ka and kd that are measured here, but on the general binding between the antibody and SpG. In fact, we found a narrow KD range between the IgGs and SpG that is likely due to the lack of interference from the V-regions present for SpA. While SpG was previously reported [With respect to SpG interactions, no notable differences in KDs  were obsreported  to bind Measurements of PpL interactions to Pertuzumab and Trastuzumab variants expectedly showed that VHs paired with Vκ1, 3 and 4 exhibits interactions as previously reported ,8. SurprAlthough the novel Vκ IgG1s bound PpL showed comparable KDs, it should be noted that the KDs were calculated from one (denoted as “+” in The unexpected IgG1 variants interacting with PpL suggested a combined VH-Vκ induced binding site to PpL that may be similar to the non-canonical binding of IgEs to nickel  in our pWith the differences between the Pertuzumab and Trastuzumab which share very similar V-regions, our findings further demonstrate the need for a design thinking  approach


Comamonas testosteroni R2 under chemostat conditions and the aerobic growth of strain R2 under batch conditions with phenol as the sole carbon source were investigated using physiological and transcriptomic techniques. Phenol-/catechol-degrading activities under chemostat conditions gradually decreased, suggesting that metabolites produced from strain R2 accumulated in the culture, which caused negative feedback. The competitive inhibition of phenol hydroxylase and catechol dioxygenase was observed in a crude extract of the supernatant collected from the collapsed culture. Transcriptomic analyses showed that genes related to nitrogen transport were up-regulated; the ammonium transporter amtB was up-regulated approximately 190-fold in the collapsed status, suggesting an increase in the concentration of ammonium in cells. The transcriptional levels of most of the genes related to gluconeogenesis, glycolysis, the pentose phosphate pathway, and the TCA and urea cycles decreased by ~0.7-fold in the stable status, whereas the activities of glutamate synthase and glutamine synthetase increased by ~2-fold. These results suggest that ammonium was assimilated into glutamate and glutamine via 2-oxoglutarate under the limited supply of carbon skeletons, whereas the synthesis of other amino acids and nucleotides was repressed by 0.6-fold. Furthermore, negative feedback appeared to cause an imbalance between carbon and nitrogen metabolism, resulting in collapse. The effects of amino acids on negative feedback were investigated. L-arginine allowed strain R2 to grow normally, even under growth-inhibiting conditions, suggesting that the imbalance was corrected by the stimulation of the urea cycle, resulting in the rescue of strain R2.The collapse of  It has been challenging to understand the principles by which microbial communities are formed , are needed. SBCs have increasing become the focus of research in recent years due to the reduced complexity of natural ecosystems and increased controllability . In our Metabolic networking systems are attracting the attention of researchers interested in the mechanisms by which microbial ecosystems are formed . In our The aims of the present study were to analyze collapsing processes using physiological and transcriptomic techniques and to develop a method that rescues strain R2 from collapse. Physiological analyses indicated that collapse was induced by feedback growth inhibition, which had already been initiated under stable conditions. The transcriptomic analysis showed that the activities of primary metabolism decreased by approximately 60 to 70% of stable conditions. An imbalance between carbon and nitrogen metabolism appeared to occur due to a decrease in the supply of carbon skeletons and an increase in ammonium influx into cells. We herein also discuss how strain R2 responded to negative feedback, the mechanisms underlying the imbalance in carbon and nitrogen metabolism in cells, and how strain R2 was rescued from collapse.C. testosteroni strain R2 was used in the present study. Strain R2 was isolated from activated sludge in wastewater treatment at an oil refinery plant (–1): 12.5 g K2HPO4, 3.8 g KH2PO4, 1.0 g (NH4)2SO4, 0.1 g MgSO4·7H2O, and 5 mL of trace-element solution (pH 7.2). The trace-element solution contained the following (L–1): 0.232 g H3BO3, 0.174 g ZnSO4·7H2O, 0.116 g Fe(NH4)2(SO4)2·6H2O, 0.096 g CoSO4·7H2O, 0.022 g (NH4)6Mo7O24·4H2O, 8 mg CuSO4·5H2O, and 8 mg MnSO4·4H2O. Cultures were harvested at the mid-exponential growth phase and then transferred to 1.5 L of BSM medium containing 0.2 mM of phenol in a chemostat reactor (capacity of 2 L). The initial cell density of strain R2 was adjusted to approximately 1.0×105 cells mL–1 by measuring optical density at 600 nm (OD600 nm). OD600 nm of 0.1 corresponded to 5.0×108 cells mL–1. After the added phenol had almost been completely degraded (the start-up phase), the chemostat culture was continuously supplied with BSM medium containing phenol  at a flow rate of 6.25 mL h–1, which corresponded to a dilution rate (D) of 0.1 day–1 . The culture volume was maintained at 1.5 L. The hydraulic residence time (HRT), calculated as 1/D, was 10 days. The culture was stirred at 150 rpm, and temperature and pH were maintained at 25°C and 7.0, respectively. Air was filtered through membrane filters with a pore size of 0.2 μm (Merck Millipore) and supplied to the culture at 1.5 L min–1. Phenol in the culture was checked using a colorimetric assay with the Phenol Test Wako kit  (g at 4°C for 5 min. The pellet was resuspended in 1 mL of 0.85% NaCI solution (w/v). The sample was incubated at room temperature for 15 min in the dark to stain cells with the LIVE/DEAD® BacLightTM bacterial viability kit L7007 (Molecular Probes) according to the manufacturer’s instructions. Samples were analyzed using the fluorescence microscope Olympus IX73 (Olympus). Stained cells were counted using ImageJ software. Cell viability was calculated as the percentage of live cells among the total cell number.The phenol-degrading bacterium g at 4°C with adequate intervals. The supernatant was sterilized by filtration through a Steriflip-GP Filter . Strain R2 was precultured in BSM medium supplemented with 2.0 mM phenol (BSM2.0phe) and 0.3 mL of filter-sterilized supernatant were transferred into 2.7 mL of fresh BSM2.0phe medium. The initial amount of cells was adjusted to OD600 nm of 0.01. As the control condition, 0.3 mL of BSM medium without phenol was added instead of the supernatant. The growth curve was automatically measured using a Bio-photorecorder . Growth parameters, including the lag time (h), growth rate constant (μ [h–1]), and amount of growth in the stationary phase (ODmax), were calculated using the growth curve. We herein defined specific growth activity as surviving activity maintaining cell density at more than 1.0×109 cells mL–1 in a chemostat culture under the condition of D. Therefore, 1 unit (U) of specific growth activity was calculated using the following equation: 1 U=0.105 (h–1)×109 (cells mL–1) under HRT of 10 days. As described above, the cell density of strain R2 was 5.0×108 cells mL–1 at an OD600 nm of 0.1. ODmax was then converted to cell density. The unit of specific growth activity was calculated according to the following equation: U=(μ×cell density from ODmax)/(1 U×lag time) (sup) to U in the control condition (Ucont) was calculated as relative growth activity (%).The effects of feedback growth inhibition on strain R2 were investigated using a supernatant collected from a pure chemostat culture and evaluated as specific growth activity. Growth curves were recorded to estimate the physiological changes that occurred after the addition of the supernatant. Strain R2 was incubated in BSM medium under the conditions of the chemostat culture supplemented with phenol as the sole carbon source. The culture was sampled and centrifuged at 5,800×The population density of strain R2 was monitored using real-time qPCR targeting the gene encoding the large subunit of phenol hydroxylase (PH). Specific sets of primers were designed by the alignment of genes encoding the large subunit of PH in strain R2  under vacuum conditions (Buchi V-700) at 40°C. The H2O fraction was mixed with methanol at a ratio of 1:3 and kept overnight at 4°C. The solution was then centrifuged at 5,800×g at 4°C and filtered  to remove precipitates. The H2O fraction was concentrated to approximately 60 mL by evaporating methanol using the rotary evaporator under vacuum conditions at 40°C. When the effects of the H2O fraction on kinetic parameters were investigated, the culture of strain R2 and the H2O fraction were mixed at a ratio of 9:1 for 30 min. Phenol- and catechol-oxygenating activities (the phenol and catechol consumption rates) were measured at various phenol and catechol concentrations, respectively, using an oxygen electrode  after respiratory oxygen consumption had been suppressed by the addition of potassium cyanide (KS (affinity constant) and Vmax  were assessed using the non-linear regression method as previously described , and the kinetic properties of strain R2 for phenol and catechol degradation were investigated using the culture on days 18, 25, 30, and 35 according to a previously described method (package ver. 3.16.4 ( genes were filtered by removing genes with low expression levels with a count per million (CPM) value of less than 2, and were normalized with scaling factors calculated for the library sizes using the Trimmed Mean of M-values (TMM) method (2 fold-change (log2FC) ≥2 (up-regulated) or ≤–2 (down-regulated) and a false discovery rate (FDR) <0.05. Raw reads for RNA-seq analyzed in the present study have been deposited in the DDBJ Sequence Read Archive (DRA) under the accession numbers DRR309243 to DRR309247 and DRR309248 to DRR209252 for samples I-1 to II-5, respectively , 22 (I-2), 30 (I-3), 31 (I-4), and 32 (I-5) from reactor I and on days 15 (sample II-1), 20 (II-2), 23 (II-3), 25 (II-4), and 28 (II-5) from reactor II (Supplementary 600 nm. A stock solution of amino acids (200 mM) was prepared in BSM medium and the filter-sterilized stock solution was added at a final concentration of 10 mM. As the control condition, 0.4 mL of BSM medium without phenol was added instead of the supernatant and amino acid solution. The growth curve was automatically measured using a Bio-photorecorder . Growth parameters, including the lag time (h), growth rate constant (μ [h–1]), and the amount of growth in the stationary phase (ODmax), were calculated using the growth curve. As described above in the section on relative growth activity, the unit of specific growth activity (U) was calculated in the presence of phenol and an amino acid as a control (Uamino acid) or in the presence of phenol, the amino acid, and the supernatant (Uamino acid+supernatant). The negative growth effect (%) was calculated using the following equation:We investigated whether the growth inhibition of strain R2 was rescued by the following amino acids: L-arginine, L-ornithine, L-citrulline, L-glutamate, and L-glutamine, because these amino acids are directly and indirectly related to the urea cycle. L-arginine, L-ornithine, and L-citrulline consisting of the urea cycle, and L-glutamate and L-glutamine are precursors for L-citrulline. Strain R2 cells precultured in BSM2.0phe liquid culture and 0.4 mL of filter-sterilized supernatant collected from collapsed chemostat cultures were transferred into 3.6 mL of fresh BSM2.0phe medium. The initial amount of cells was adjusted to 0.01 at ODamino acidU⁄amino acid+supernatantU)×100.(1–C. testosteroni strain R2 on days 18 and 38 in the chemostat culture. Samples were fixed with a mixed solution of 25% (v/v) glutaraldehyde and 5% (v/v) formaldehyde for 2 h in potassium phosphate buffer  and dehydrated with ethanol with serially increasing concentrations  and then with 100% of butyl alcohol. The sample was mounted on an aluminium stub with double-sided carbon type, and sputter-coated with gold under argon at a thickness of 50 Å in the Quick Auto Coater  for 20 s. Following coating, samples were imaged at different magnifications with a field emission scanning electron microscope  at an acceleration voltage of 5 kV and working distance of 5 mm.SEM was used to observe the morphology of –1, and elutes were monitored at 210 nm. Phenol and catechol were identified according to their retention times of 7.2 and 5.2 min, respectively. Concentrations were assessed by comparing the peak area with that of the cognate standard sample.Phenol and catechol concentrations were monitored using high-pressure liquid chromatography (Waters Japan) equipped with a column  and UV detector. Liquid samples collected from the chemostat culture were centrifuged and filtered . Liquid samples were eluted using 50% acetonitrile solution with 20 mM ammonium acetate delivered at 0.1 mL mint-test. P=0.05 was considered to be significant.DEGs were identified by the likelihood-ratio test implemented in the edgeR package. Other data were analyzed using the Student’s 600 nm from days 4 to 14 and then gradually decreased  and found that the H2O fraction exerted the strongest negative effects on the relative growth activity of strain R2  collected from chemostat cultures of strain R2 on kinetic parameters for phenol and catechol degradation. As a preliminary test, we fractionated the supernatant using Hfraction C and D.ca. 41–54 million quality-filtered transcript reads per sample  and nitrate/nitrite transport system (CTR2_4163) was ~200-fold higher in the collapsed status (I-5 and II-5) than in the stable status (I-1 and II-1). On the other hand, common functional features were not observed among the down-regulated genes in reactors I and II  of strain R2 under the same conditions and performed comparative transcriptomics. The population densities and phenol concentrations of strain R2 in chemostat reactors I and II slightly differed, but showed similar changes  synthesis  and catechol 2,3-dioxygenase . The genes encoding PH and catechol 2,3-dioxygenase were up-regulated by approximately 30- and 3.3-fold, respectively, in reactor I, and by 15- and 1.2-fold, respectively, in reactor II  significantly decreased by 0.34±0.065-fold in the collapsed status , and to 0.80±0.23-fold from succinate to oxaloacetate    fluctuated in the collapsed status and became similar to or higher than those in the stable status.In the TCA cycle, the relative transcriptional levels of genes encoding enzymes related to the conversion of 2-oxoglutarate (2-OG) to succinate  . In the R2_1688) . The relgdhA, CTR2_3645) in the collapsed status decreased to ~0.40-fold those in the stable status. The relative transcriptional levels of glutamate synthase (GOGAT)  and glutamine synthetase (GS)  increased by approximately 2-fold in the collapsed status  moderately decreased to 0.82±0.22-fold in the collapsed status, whereas those of genes encoding enzymes related to the conversion from L-ornithine to L-arginino succinate via L-citrulline  significantly decreased to 0.47±0.051-fold in the collapsed status , L-glutamate, and L-glutamine . Lag tim control . Growth  control . ODmax iobserved B. Negatiobserved , even whThe present study attempted to elucidate the collapsing mechanism induced by feedback growth inhibition in strain R2. Collapse was not observed under batch conditions, it only occurred under chemostat conditions. The results obtained revealed that growth inhibition and metabolic changes had already occurred before the collapse, which was attributed to negative feedback induced by the accumulation of hydrophilic metabolites produced from strain R2. Unexpectedly, increases were noted in the relative transcriptional levels of the genes encoding PH and catechol 2,3-dioxygenase , Table 1aceA, CTR2_1688) related to the TCA cycle and glyoxylate shunt fluctuated and increased under collapsed conditions  decreased  and GOGAT  (the GS-GOGAT pathway) increased  and the urea ABC transporter (CTR2_0055) were up-regulated by ~155- and ~20-fold, respectively, in the collapsed status  and cell division protein (CTR2_0482) were down-regulated  Imbalance in Carbon and Nitrogen Metabolism in https://doi.org/10.1264/jsme2.ME21050Supplementary Material


To detect and investigate the expression of POU domain class 2 transcription factor 2 (POU2F2) in human lung cancer tissues, its role in lung cancer progression, and the potential mechanisms.Immunohistochemical (IHC) assays were conducted to assess the expression of POU2F2 in human lung cancer tissues. Immunoblot assays were performed to assess the expression levels of POU2F2 in human lung cancer tissues and cell lines. CCK-8, colony formation, and transwell-migration/invasion assays were conducted to detect the effects of POU2F2 and AGO1 on the proliferaion and motility of A549 and H1299 cells in vitro. CHIP and luciferase assays were performed for the mechanism study. A tumor xenotransplantation model was used to detect the effects of POU2F2 on tumor growth in vivo.We found POU2F2 was highly expressed in human lung cancer tissues and cell lines, and associated with the lung cancer patients’ prognosis and clinical features. POU2F2 promoted the proliferation, and motility of lung cancer cells via targeting AGO1 in vitro. Additionally, POU2F2 promoted tumor growth of lung cancer cells via AGO1 in vivo.We found POU2F2 was highly expressed in lung cancer cells and confirmed the involvement of POU2F2 in lung cancer progression, and thought POU2F2 could act as a potential therapeutic target for lung cancer.The online version contains supplementary material available at 10.1186/s12890-021-01476-9. Lung cancer often originates from bronchial mucosa or gland, and is a malignant tumor with relative high mortality , 2. In 2POU domain class 2 transcription factor 2 (POU2F2), also named octamer-binding protein 2 (OCT2), was a B-cell-restricted transcription factor belonged to POU domain family, which used the POU domain to bind DNA , 10. It Recently, the critical role of POU2F2 in the progression of cancers has been revealed in depth . POU2F2 Herein, we found the high POU2F2 expression in human lung cancer tissues and cells. POU2F2 expression was correlated with patients’ prognosis. We also found POU2F2 could promote the proliferation, and motility of lung cancer cells via AGO1, and contribute to tumor growth of lung cancer cells in mice. We therefore thought POU2F2 could act as a promising target for lung cancer.Anti-POU2F2 , 1:1000 for Immunoblot, and 1:50 for CHIP, ab179808, Abcam, UK), anti-AGO1 , anti-Ki67 , anti-PCNA , anti-N-cadherin , and anti-β-actin . POU2F2 and AGO1 siRNAs were bought from Riobio Biotech. The shRNA of POU2F2, plv-vector, plv-POU2F2, plv-AGO1, and pGL3-Basic plasmids was purchased from Addgene plc.The lung cancer tissues and the adjacent tissues were all obtained from The Fujian Medical University. IHC assays were performed on these tissues. Resin was used for the embedding of tissues, and 5-µm-thick sections were fixed with the 4% paraformaldehyde (PFA) for half an hour at room temperature. Subsequently, sections were blocked with 2% BSA  for half an hour at room temperature. Sections were treated with the antibody for 2 h at room temperature. Then the sections were treated with the secondary antibodies for 1.5 h, and the diaminobenzidine was used as a type of chromogen substrate. A light microscope was used for imaging at 100 × and 200 × magnification. All procedures performed in studies involving human participants were in accordance with the standards upheld by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University and with those of the 1964 Helsinki Declaration and its later amendments for ethical research involving human subjects.2 incubator.4 different types of human lung cancer cell lines, calu-3, calu-6, A549, and H1299, and a normal bronchial epithelial cell line, named BEAS-2B, were bought from ATCC and maintained in dulbecco’s minimum essential medium (DMEM), supplemented with 10% of fetal bovine serum (FBS) and incubated at 37 °C in a 5% COThe plasmids or siRNAs were transfected into lung cancer cells by lipofectamine 3000 . The in vivo assays used Stable POU2F2 depletion cells were identifed by POU2F2 shRNA transfection.Protein samples were isolated by SDS-PAGE, transferred onto the NC membranes, followed by 5% fat-free milk blocking. NC membranes were subsequently incubated with POU2F2, AGO1, and β-actin antibodies for 1.5 h. Subsequently the membranes were incubated with HRP-labeled secondary antibodies for an hour. Signals were detected by ECL kit . These images were analyzed with the Image J .For CCK-8 assays, both A549 and H1299 cells (800 cells/well) were added into 96-well plates for 3 days. CCK-8 agent was added into the plate for 3 h at 37 ℃. Then the OD value in each well was detected at 490 nm wavelength.For colony formation assays, cells were plated into 24-well plates with a density of nearly 100 cells perwell and maintained for 2 weeks. The colonies were fixed with methanol at – 20 ℃ for half an hour and stained with 0.1% crystal violet for half an hour. Colonies were then counted.Lung cancer cells were transfected, trypsinized, and re-suspended in medium without serum.For transwell-invasion assay, the upper chambers of 8.0 µm membrane pore transwell filters coated with 20% matrigel (For transwell-migration assay without matrigel) and incubated at 37 °C for half an hour.Then lung cancer cells in 150 µL medium were all added to the upper chambers to stimulate the migration toward the bottom containing complete medium. After 24 h, the remaining cells in the top chamber were removed, and the remaining lung cancer cells were fixed with 4% paraformaldehyde (PFA), followed by the staining of 0.1% crystal violet, and the OD value at 490 nm wavelength was measured.To measure tumor volume, A549 cells stably transfected with the shRNA plasmids were injected subcutaneously into the nude mice. After 1 week, tumors were established, and the volume of tumors was measured every week and calculated. After 7 weeks, all tumors were isolated, and tumor growth curves were calculated respectively, and the weight of tumors were also measured. All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University.CHIP assays wereconducted by a CHIP assay kit . A total of 108 A549 cells were crosslinked, mixed, and lysed. Then the samples were sonicated to shear the DNA into between 600 and 800 bp. Chromation fraction was isolated by the anti-POU2F2 or IgG antibodies. Then, the mix was enriched by the agarose of protein A/G, which were subsequently isolated and washed. DNA was purified, and the qPCR assays were subsequently performed to detect site 1, site 2 and site 3 level.For luciferase assays, A549 cells were transfected with plv-vector or plv-POU2F2 plasmids overnight. After 24 h, cells were lyzed, and the of AGO1 wild or mutant cell luciferase activities were detected by a luciferase assay kit.t test was for statistical comparisons, and p < 0.05 was considered significant. *indicated p < 0.05, **p < 0.01, and ##p < 0.001, respectively.In this study, graphpad 6.0 software was used for the statistical analysis. All data were represented as mean ± SEM. K-M survival analysis was used to assess the survival of patients. Student’s To investigate the potential role of POU2F2 in the progression of lung cancer, we first assessed the expression levels of POU2F2 in lung cancer tissues and the adjacent tissues isolated from patients through IHC assays. Importantly, we noticed the high expression levels of POU2F2 in human lung cancer tissues, compared to normal tissues Fig. a. FurtheWe next explored whether POU2F2 was highly expressed in human lung cancer cell lines compared to normal bronchial epithelial cell lines. Four types of human lung cancer cell lines, including calu-3, calu-6, A549, and H1299, and a normal bronchial epithelial cell line BEAS-2B were used to detect the expression of POU2F2 in these cell lines via Immunoblot assays. Similarly, to the previous results, we noticed POU2F2 was upregulated in human lung cancer cell lines compared to the bronchial epithelial cell line , age (p = 0.370), histology (p = 0.777), and differentiation (p = 0.395). Importantly, we found the expression of POU2F2 was obviously correlated with the tumor size (p = 0.021), TNM stage (p = 0.024), and nodal status (p = 0.047) of patients with lung cancer  or low expression  groups, according to the staining intensity of POU2F2 Table . We founp = 0.0385), suggesting the obvious correlation with prognosis , we found POU2F2 could target AGO1 promoter regions, including three different sites  . AGO1 coIn summary, we noticed the high POU2F2 levels in human lung cancer tissues and cell lines. POU2F2 expression correlated with the prognosis and clinical features of lung cancer patients. We further demonstrated POU2F2 could promote the proliferation and motility of lung cancer cells through transcriptionally activating AGO1, and fascinate tumor growth of lung cancer cells in mice. We therefore thought POU2F2 as a lung cancer target.Additional file 1. The original WB image in figure1B.Additional file 2. The original WB image in figure1C.Additional file 3. The original WB image in figure 2A.Additional file 4. The original WB image in figure 3A.Additional file 5. The original WB image in figure 4B.Additional file 6. The original WB image in figure 5B.Additional file 7. The original WB image in figure 6A.Additional file 8. The original WB image in figure 7B.