Tuning is facilitated by the changesin orbital hybridization that favor different superexchange pathways.This can produce alternative magnetic structures when B″ isd10 or d0. Furthermore, the competition generatedby introducing mixtures of d10 and d0 cationscan drive the material into the realms of exotic quantum magnetism.Here, Te6+ d10 was substituted by W6+ d0 in the hexagonal perovskite Ba2CuTeO6, which possesses a spin ladder geometry of Cu2+ cations, creating a Ba2CuTex1–WxO6 solid solution(x = 0–0.3). We find W6+ is almostexclusively substituted for Te6+ on the corner-sharingsite within the spin ladder, in preference to the face-sharing sitebetween ladders. The site-selective doping directly tunes the intraladder, Jrung and Jleg, interactions.Modeling the magnetic susceptibility data shows the d0 orbitalsmodify the relative intraladder interaction strength (Jrung/Jleg) so the system changesfrom a spin ladder to isolated spin chains as W6+ increases.This further demonstrates the utility of d10 and d0 dopants as a tool for tuning magnetic interactions in a widerange of perovskites and perovskite-derived structures.Isovalentnonmagnetic d 10 or d0 cationsmodifies the magnetic interactions in perovskites due to their differingcontributions to superexchange. Here, we demonstrate the d10/d0 effect can be site-selectively applied to the corner-sharingsite in a hexagonal perovskite, Ba2CuTex1−WxO6. Thisdirectly tunes the intraladder interactions in the Cu2+ spin ladder and modifies the magnetic behavior. This further demonstratesthe utility of the d10/d0 effect for modifyingmagnetic interactions in perovskites.Substitution of d The periodic table offers a plethoraof dopants from which to choose on the basis of differences in chargeand ionic radii. By careful selection, it is possible to desirablymodify the structural, electronic, and magnetic properties and, insome cases, to generate behaviors entirely different from those ofthe parent compound. The classic example is Sr4 However, the situation is more complicated whenthe magnetic cations are farther away and the interactions occur byextended superexchange. Recently, a new method for directly tuningthese extended superexchange interactions has been developed.6 This method is based on doping diamagnetic d10 and d0 cations into extended superexchange pathways that link magneticcations. This d10/d0 effect can be used in A2B′B″O6 double perovskites, whereB′ is a magnetic cation and B″ is a diamagnetic d10 or d0 cation.8 The doubleperovskite structure consists of corner-sharing B′O6 and B″O6 octahedra alternating in a rock salt-typeorder .Dopants have such dramatic effects because they intrinsically modifythe interactions within the parent material. In magnetic oxides, theseinteractions are typically superexchange interactions mediated byoxygen anions. These interactions are generally well understood whenthe magnetic cations are connected by a single oxygen anion.peorder 1a.9 The 10 and d0 cations on the linkingB″ site have a significanteffect on the magnetic interactions and ground states in double perovskites.10 We investigated this d10/d0 effect in thecubic double perovskites Ba2MnTeO6 and Ba2MnWO6, in which the magnetic Mn2+ cationsare linked by either 4d10 Te6+ or 5d0 W6+ B″ cations. In these isostructural materials,Mn2+S = 5/2 magnetismis described using a simple face-centered cubic (fcc) Heisenberg modelconsisting of a 90°  and 180°  Mn–O–(Te/W)–O–Mninteraction (J1 when B″ = Te6+ (4d10) and a stronger J2 when B″ =W6+ (5d0). The contrasting J1 and J2 interactions produceentirely different magnetic structures for Ba2MnTeO6 (type I AFM) and Ba2MnWO6 (type IIAFM).We have recently shownthat diamagnetic deraction 1a. Neutr10/d0 effect is caused by differencesin orbital hybridization in the B′–O–B″–O–B′superexchange pathways. When B″ = Te6+, there isno d-orbital contribution to superexchange as the 4d10 orbitalslie far below the Fermi level.11 Therefore,the majority of superexchange occurs via a NN B′–O–O–B′interaction.13 Conversely, when B″ =W6+, the 5d0 orbitals strongly hybridize withO 2p allowing W6+ to directly contribute to extended superexchangevia NNN B′–O–W6+–O–B′.14 This effect limits the NNN J2 exchange in Te6+ compounds, as this superexchangepathway requires a d-orbital contribution from the B″ cation.We also highlight the fact that the d10/d0 effectextends beyond simple cubic structures to a large range of 3d transitionmetal B′ = Co,16 Ni,19 and Cu21 double perovskites, all of which follow the sameprinciple based on the nonmagnetic B″ site: d0 withstrong J2 (type II) or d10 withstrong J1 (type I/Néel order).The d10/d0 effectin 3d double perovskites are the Cu2+S = 1/2 compounds Sr2CuTeO6 and Sr2CuWO6 and their solid solution Sr2CuTex1–WxO6, where the d10/d0 dopingstabilizes a novel quantum disordered ground state. Here, the combinationof the Cu2+ Jahn–Teller (J–T) effect andorbital ordering produces a square lattice Heisenberg antiferromagnet,with highly two-dimensional magnetism.22 The tetragonal unit cell has square lattice a–b planes of Cu2+ cations in which superexchangeis described using in-plane J1 (NN) and J2 (NNN) interactions, but with additional weakinterplane interactions (J3 and J4) along c  and strong J2 (x = 1) limits.26 The d10/d0 substitution results in the strong suppressionof magnetic order as a quantum disordered ground state is observedfor a wide composition range of x = 0.05–0.6.29 The 50:50 mixture Sr2CuTe0.5W0.5O6 closely resemblesa quantum spin liquid, an exotic magnetic state in which the momentsremain dynamic at 0 K and have been highly sought since they werefirst proposed in the 1970s.33The most striking examples of the d along c 1b.11,21 10/d0 doping canbe used to tune magnetic interactions and induce exotic magnetic statesin other magnetic lattices than the square lattice remains, and whetherthis can be extended from perovskites to perovskite-derived structures.Depending on the choice of A and B′/B″ cations, B′–O–B″–O–B′linkers form between corner-sharing or/and face-sharing octahedra,generating the classic double perovskite structure in the purely corner-sharingcase, while the introduction of face sharing creates the hexagonalperovskite structure.36 The observation of tunable magnetic interactionsin structures with different octahedral connectivity would suggestd10/d0 substitutions can be employed in a rangeof materials to access novel quantum states, many of which are hardto realize experimentally.37The question of whether d2CuTeO6 is an excellent system for testingthis due to its hexagonal perovskite structure that results in a spinladder magnetic geometry.41 Within the spin ladder, Cu2+ cations arelinked via three key Cu–O–Te–O–Cu exchangeinteractions illustrated in Jleg and Jrung interactions viathe corner-sharing Te(1)O6 units and the interladder interactionvia the face-sharing Te(2)O6 units within the Cu–Te(2)–Cutrimers.42 The intraladder interactionsare antiferromagnetic and equally strong with Jrung/Jleg ∼ 1, while theinterladder interaction is weaker.39 In principle,W6+ could be doped onto either of the Te6+ B″sites. This offers the possibility of tuning the Jleg and Jrung interactionsindependently of the Jinter, forming amore complex phase space than cubic perovskites. For clarity, thetwo B″ sites are henceforth labeled B″(c) and B″(f),where c and f denote corner and face sharing, respectively. The B″(c)and B″(f) sites are indicated in 2+ spinladders running along the b-axis of the Ba2CuTeO6 structure. The intraladder interactions in Ba2CuTeO6 are quite similar to the Cu–O–Te–O–Cuinteractions within the square lattice of Sr2CuTeO6. Both structures share the structural motif shown in 2+ cations interacting via Cu–O–B″–O–Cusuperexchange. In addition, the significant Jinter leads to the formation of a Néel ordered groundstate for Ba2CuTeO6, the same type of orderingobserved for Sr2CuTeO6.39 Hence, in a manner analogous to that of Sr2CuTex1–WxO6, onemight expect similar strong suppression of magnetic order upon site-specificdoping of B″ d0 cations onto the intraladder B″(c)sites in Ba2CuTex1–WxO6.29Ba2CuTex1–WxO6 solid solution (0 ≤ x ≤ 0.3).Using a combination of crystallographic and spectroscopic techniques,we show that W6+ can be site-selectively doped onto thecorner-sharing B″(c) site in Ba2CuTex1–WxO6. Thissite selectivity allows for the direct tuning of intraladder interactions,which show a strong decrease in Jrung/Jleg with an increase in x.Our work demonstrates the d10/d0 effect canbe extended to perovskite-derived structures such as hexagonal perovskites.To answer these questions, we prepared theBa22.12CuTex1–WxO6. The x = 0, 0.05,0.1, 0.2, and 0.3 compositions were prepared by thoroughly mixingstochiometric quantities of high-purity BaCO3 (99.997%),CuO (99.9995%), TeO2 (99.995%), and WO3 (99.998%) in an agate mortar. The reactant mixtureswere pelletized and calcined at 900 °C in air, before being firedat 1000–1100 °C for 24 h periods with intermittent grinding.The phase purity was monitored using X-ray diffraction . A total of 72–120 h was required to achieve phasepurity in all compositions, with the heating time increasing as theW content increased. The synthesis was stopped once single-phase sampleswere obtained. The sample color changed from yellow to a darker yellow-brownacross the solution from x = 0 to x = 0.3, which may be indicative of a gradual modification of theband gap as the W6+ content increased.Conventional solid-statechemistry techniques were used to synthesize polycrystalline samplesof Ba2.22CuTex1–WxO6:Ag powder was pressed into a pellet. The pellet was broken,and ∼10 mg shards were selected; the heat capacity was measuredbetween 2 and 120 K using the thermal relaxation method. The silvercontribution was removed on the basis of a measurement of pure silverpowder.Magneticcharacterization was performed using a Quantum DesignMPMS3 magnetometer (Magnetic Property Measurement System). Approximately100 mg of powder was sealed in a gelatin capsule, which was then securedin a polymer straw sample holder. Zero-field-cooled (ZFC) and field-cooled(FC) curves were measured between 2 and 300 K in dc superconductingquantum interference device mode using an external field of 0.1 T.ac measurements were taken between 2 and 100 K using a dc field of25 Oe and an ac field of 5 Oe using ac frequencies between 10 and467 Hz. Heat capacity measurements were performed using a QuantumDesign Physical Property Measurement System instrument. The sampleswere mixed with silver (99.999%) in a 1:1 gravimetric ratio to enhancethe low-temperature thermal conductivity. The Ba2.3x = 0.05, 0.1, and 0.3 compounds wasinvestigated using the High Resolution Powder Diffractometer (HRPD)at the ISIS Neutron and Muon Source. Approximately 8 g of each samplepowder was loaded into an Al-alloy slab-can and sealed using vanadiumwindows. The exposed surfaces of the slab-can were shielded usinghighly absorbing Gd and Cd foils so that only the vanadium windowsof the can were exposed to the neutron flux. After the slab-can hadbeen aligned perpendicular to the neutron beam, time-of-flight neutronpowder diffraction patterns were recorded between 2 and 300 K usinga cryostat to cool the sample. The data can be found online.43 The data were corrected for sample absorption,and Rietveld refinements were performed using GSAS-2.45 VESTA was used to visualize the crystal structures.46The nuclearstructure of 2.4x = 0.1, 0.2, and 0.3 samples were loadedinto 0.6 mm diameterglass capillaries and measured at room temperature on the P02.1 beamlineat the PETRA III X-ray radiation source (DESY) using a wavelengthλ of 0.20742 Å. The capillary was located 1169.45 mm fromthe PerkinElmer XRD1621 two-dimensional (2D) detector and spun duringthe measurement. The 2D data were processed using DAWN Science. Theone-dimensional data obtained were refined using GSAS-2. The X-rayand neutron data (as well as the bulk magnetization data) were collectedusing samples from the same batch.The 2.5x = 0.3 compound were recorded in transmission mode near the W L3 edge, using a finely ground specimen dispersed in polyethyleneglycol to achieve a thickness of one absorption length. Incident andtransmitted beam intensities were measured using ionization chambers,filled with mixtures of He and N2, operated in a stableregion of their I/V curve. A tungstenfoil was used as an internal energy calibration where the first inflectionpoint in the measured W L3 edge was defined to be E0 = 10206.8 eV. Data reduction and analysiswere performed using Athena, Artemis, and Hephaestus.47 The EXAFS data were then analyzed using ATOMS and FEFFin the Artemis package with the monoclinic structural model.EXAFS measurementswere performed on the Beamline for MaterialsMeasurement (6-BM) at National Synchrotron Light Source II (NSLS-II).Room-temperature X-ray absorption spectra (XAS) of the 33.1x = 0 to x = 0.3. Attemptswere made to synthesize richer W6+ samples, but beyond x = 0.3, significant W6+ impurities formed. Theseimpurities were not diminished on further heating, showing the x = 0.3 composition lies close to the solubility limit forour synthesis procedure. Rietveld refinement showed all of the Ba2CuTex1–WxO6 structures adopt the same C2/m symmetry as the Ba2CuTeO6 parentstructure. The unit cell volume decreases linearly with x ; this showingVegard’s law behavior indicates successful doping of W6+ into the Ba2CuTeO6 structure. SynchrotronX-ray diffraction, EXAFS, and neutron diffraction studies providedfurther insight into the structural effects of doping across the solution.Initial structuralcharacterization was achieved using laboratory X-ray diffraction.The laboratory X-ray diffraction (XRD) patterns confirm phase purityfrom 3.1.1x = 0.2 sample. Data collected at 300 K for x = 0.1,0.2, and 0.3 compounds were all used to test three possible site occupancymodels. These models are (1) W6+ exclusively on the B″(c)site, (2) W6+ exclusively on the B″(f) site, and(3) W6+ occupying both B″(c) and B″(f) sites.An equal distribution (50:50) was initially assumed in model 3. Theresults in panels a and b of Rwp is consistently lower whenW6+ exclusively occupies the B″(c) site in x = 0.1, 0.2, and 0.3 compounds. This suggests a strongpreference for corner-sharing, which was further evaluated by allowingthe site occupancies to be refined, within the constraints of samplestoichiometry. This identified a small amount of W6+ onthe B″(f) site in each sample, with the site occupancy increasinglinearly with x to a maximum value of ∼3%as shown in 6+ present in the sampleis found on the face-sharing octahedral site. Comparing the Rwp and χ2 values in 6+ leads to a slight but notnegligible improvement in the fit compared to model 1. W6+ occupancy of the B″(f) site was confirmed by refinementsusing the initial site occupancies from model 1 and model 2 as startingvalues. Both refinements converged to the same results in 3.1.23 EXAFS data consideredthe following models, (1) full W6+ substitution on theB″(c) site and (2) full W6+ substitution on theB″(f) site, within the monoclinic structure. Model 1 affordeda plausible W6+ environment at the B″(c) site, withreasonable path lengths and positive Debye–Waller factors (Table S11). R-factor of 1.18%. In contrast, model 2 did notafford a plausible W6+ environment at the B″(f)site, with several paths having negative Debye–Waller factors(Table S12). The fit for model 2 in R-factor of 9.07%. This is because W6+ substitutionat the B″(f) site does not provide adequate scattering pathsto fit the significant second-shell contribution observed in the χ(R) transform of the EXAFS data in the R range of 3–4 Å . This supports the strong preference for W6+ doping at the B″(c) site, in agreement with the synchrotronX-ray data, but has the added advantage of providing an element-specificperspective. Attempts were made to fit the EXAFS data using contributionsfrom both models 1 and 2, under a linear constraint, to assess thepotential for disorder of a fraction of W6+ from the B″(c)to B″(f) site. However, it was not possible to adequately stabilizesuch a fit, because the number of variables approached the numberof data points.Analysis of W L3.1.349 Consequently, variable-temperatureneutron diffraction studies were performed on x =0.1 and x = 0.3 compounds between 2 and 300 K toidentify the low-temperature structure across the series. Both x = 0.1 and x = 0.3 compounds undergo thesame C2/m to P1 transition as Ba2CuTeO6 on cooling.42 The transition Ttrans was marked by peak splitting, as one can observe by comparing theneutron diffraction patterns of Ba2CuTe0.7W0.3O6 at (a) 300 K and (b) 1.44 K in x inBa2CuTex1–WxO6 increased, peak splitting was suppressedto lower temperatures. Where Ttrans isjust below room temperature (287 K) for Ba2CuTeO6,42 comparing the R-factorsfrom refinements using the C2/m and P1 models places Ttrans between 235 and 240 K in the x =0.1 compound and 100–120 K in the x = 0.3compound. The decrease in Ttrans is expectedto follow across the series down to the minimum at x = 0.3 as the level of cation disorder increases with x. Importantly, it is clear the low-temperature structure at 2 K istriclinic.In low-dimensionalsystems, the most striking quantum magnetic behavior may emerge atlow temperatures.C2/m to P1 is caused by weak symmetry breakingarising from further J–T distortion of the CuO6 octahedraon cooling. The structural integrity of the 12R hexagonal stackingsequence is retained upon the transition to lower symmetry. Like inthe C2/m structure, extended Cu–O–B″–O–Cusuperexchange occurs via the corner-sharing B″(c)O6 and face-sharing B″(f)O6 units forming the intraladderand interladder exchange interactions depicted in C2/m structure has been previously usedto model the low-temperature magnetic interactions as the higher symmetrysimplifies the calculations.39The transition from 6 octahedrawas measured empirically using the J–T distortion parameter(σJT) in 51i represents the Cu–O neutron bondlength on the i-Cu(1)O6 site and ⟨Cu–O⟩is the mean bond length. σJT isplotted as a function of T for x = 0.1 and x = 0.3 compounds in Figure S15. As expected, σJT was found to be large and non-zero, reflecting uneven elongationof the axial Cu–O bonds to accommodate both corner and facesharing with the B″O6 octahedra. σJT gradually increased with a decrease in temperaturefor both samples, with the increasing distortion driving the transitionto P1 symmetry. It appears theCuO6 octahedra in the x = 0.3 sample areslightly less distorted, possibly due to differences in covalencybetween W6+ and Te6+. However, compared to x = 0.1, the difference in σJT is minor and both samples plateau to the same distortionlimit at 100 K.Thedependence of temperature on the CuOThe similarity in the neutron scattering lengthsof Te and W, andthe similarity of the preferred coordination site of these cations,meant it was not possible to determine the B″(c) versus B″(f)site distribution from the neutron diffraction data. Therefore, theB″(c) and B″(f) site occupancies determined from thesynchrotron X-ray diffraction data were used in the crystal structuralmodels.3.23.2.1x = 0, 0.05, 0.1,0.2, and 0.3 compounds using a field of 0.1 T are shown in T of x = 0 is identical toprevious reports.40 Upon cooling, there is a broadmaximum at a Tmax of ∼74 K, correspondingto short-range ladder interactions. Below Tmax, the susceptibility decreases before leading onto a small upturnbeyond 14 K. The low-temperature behavior is believed to be indicativeof the departure from ladder behavior and entry to the long-rangeordered Néel state.38 However, thelow-dimensional magnetic behavior means the system does not presenta classical AFM ordering cusp, and the low-temperature upturn is nota general sign of magnetic order. Instead, magnetic order has beendetected using muon and inelastic neutron scattering techniques placingthe magnetic transition at a TN of 14K.41 It is likely to be coincidental that theupturn occurs close to the TN of Ba2CuTeO6.dc magneticsusceptibility data collected for 6+-doped samples are similar in that they all displaythe same broad Tmax feature, but the positionof Tmax shifts to lower temperatures as x increases as shown in x creating a “Curie-tail”-likefeature that is most pronounced for the x = 0.3 sample.These Curie-tail features show no field dependence upon measurementat higher external fields of 1 T.The W2CuTex1–WxO6 given the full Te/W disorder on the B″(c)site. We did not observe any ZFC/FC divergence in the dc magneticsusceptibility for any of the samples. We further investigated thepossibility of a spin glass state by measuring the ac magnetic susceptibilityof the x = 0.1 and x = 0.3 samples,shown in panels c and d of χ′ac versus T data for each of these samplesshow no frequency-dependent shift in the curve between 2 and 100 K.Furthermore, no peaks were detected in the imaginary component (χ″ac vs T) of theac susceptibility. As such, we find no evidence supporting a spinglass state in Ba2CuTex1–WxO6.A spin glassstate might be an expected ground state of Ba2CuTe0.9W0.1O6 in C) and Weiss temperatures (θW)for each sample are listed in W across the series, implying the interaction strength remainsconstant. The value of C was used to calculate theeffective magnetic moment μeff. The value of μeff is ∼2 μB per Cu2+ foreach sample, larger than expected for a Cu2+ moment, butclose to the previously reported value of 1.96 μB per Cu2+ for Cu2+ in Ba2CuTeO6.38 It is unclear why the effectivemagnetic moment is enhanced in these samples.The dc magneticsusceptibility data between 200 and 400 K werefitted using the Curie–Weiss law as illustrated for BaT data were fitted using anisolated two-leg spin ladder model.52 Thismodel is based on highly accurate Quantum Monte Carlo (QMC) calculationsand allows us to investigate how the intraladder interactions aremodified by W6+ substitution. The model has three fittingparameters: the main interaction Jleg,the ratio Jrung/Jleg, and g. The isolated spin ladder modelwas previously used to show that intraladder interactions are equallystrong in Ba2CuTeO6 (Jrung/Jleg ∼ 1),41 which was in excellent agreement with density functional theorycalculations38 and subsequent inelasticneutron scattering measurements.39x = 0 are in excellent agreement with previous literature.41 Upon W6+ substitution, the strength of the main Jleg interaction is relatively stable showinga small increase. However, the relative strength of the intraladderinteractions changes significantly as x increases.The Jrung/Jleg ratio decreases from near unity  for x = 0, to a value of Jrung/Jleg = 0.11(14) upon reaching x = 0.3. This shows that W6+ doping leads to a strong suppressionof the Jrung interaction.The χ versus Jleg has twice the impactof Jrung, because Jleg connectsany Cu2+ site to two neighboring sites while Jrung connects to only one. Thus, the effect of the smallincrease in Jleg and the strong suppressionof Jrung is a moderate weakening of theoverall interactions. This is consistent with the shift in Tmax to lower temperatures, but not with ourrelatively constant trend in θW obtained from Curie–Weissfits. The reason for this discrepancy is not known, but it could berelated to the strong quantum fluctuations arising from the nearbyquantum critical point in Ba2CuTeO6.39In terms of the overall strength of the magneticinteractions, Jleg and Jrung interactions and more spin chain-type systemswith suppressed Jrung are not immediatelyobvious. The isolated spin ladder fitting function used here is basedon QMC calculations of the magnetic susceptibility for different Jrung/Jleg ratios.52 The QMC calculations show that differences inthe high-temperature susceptibility are minor between an isotropic Jrung/Jleg = 1 spinladder and a spin chain with the latter showing a small increase.The main difference between the two models is found in the broad maximumat Tmax. For isotropic Jrung/Jleg = 1 ladders, thissusceptibility is relatively sharp and highly asymmetric. As the Jrung/Jleg ratiodecreases, the maximum becomes both broader and much more symmetric,while shifting to lower temperatures. We observe these expected trendsin our Ba2CuTex1–WxO6 samples: the maximum becomesbroader and more symmetric with an increase in x whilealso shifting to lower temperatures. This further shows that the Jrung/Jleg ratiodoes decrease with W6+ doping. The differences in the broadmaximum are highlighted in x = 0 and x = 0.2. It shouldbe noted that the Curie-like feature observed at low temperaturesis too small to explain the changes in the broad maximum.The differences in magnetic susceptibilitybetween an isotropicspin ladder with equally strong Jrung/Jleg decreaseswith an increase in x in Ba2CuTex1–WxO6, we comparethe best fits froman isotropic spin ladder model52 (Jrung/Jleg = 1) anda spin chain model54 (Jrung/Jleg = 0) for x = 0and x = 0.2 compounds in panels b and c of x = 0 very well, and the Jrung/Jleg ratioof ∼1 has been confirmed by density functional theory calculationand inelastic neutron scattering.39 As expectedfor the x = 0 compound, the spin chain model providesa poor fit for the maximum in susceptibility, but also for the high-temperaturesusceptibility. In contrast, the isotropic spin ladder model providesa poor fit for the x = 0.2 data, especially for thebroad maximum. The spin chain model, however, describes the x = 0.2 data well and provides a noticeably better fit thanthe isotropic spin ladder model for the maximum but also at high temperatures.This confirms W6+ doping in Ba2CuTex1–WxO6 changesthe relative strength of magnetic interactions by decreasing the Jrung/Jleg ratio.Spin chain fits to all compounds are presented in Figure S29, and this model provides a progressively betterfit to susceptibility data with an increase in x.To conclusively show that 3.2.2PC) measurements were performed on allsamples. The plot in PC/T data as a function of T. Close examination of thecurves shows no evidence of an ordering transition in the parent ordoped samples. The former observation agrees with previous heat capacitymeasurements on Ba2CuTeO6.40 Because of weak Néel ordering, Ba2CuTeO6 possesses strong quantum fluctuations that spread out the magneticentropy. Hence, PC/T measurementsare largely insensitive to any trace of a λ ordering peak about TN. The lack of a λ peak in the x > 0 curves shows magnetism in the doped samples issimilarlyweak, as expected from the small S = 1/2 Cu2+ moment and low-dimensional behavior.Consequently, the curves appear to be much the same, so it is notpossible to distinguish differences in magnetic ordering. The smallvariation in the high-temperature data is an artifact of the silvercontribution to PC/T,which is very well corrected for at low temperatures. However, thereare notable trends in the low-temperature PC/T data. The expansion of the range of 2–10K in PC/T and T2 that is readily fittedusing the Debye–Einstein model (PC = γT + βDT3). The electronic contribution (γ) to the heatcapacity was extracted from the intercept of PC/T versus T2 and plotted as a function of x in x = 0 sample, the value of γ is almost zero, in excellent agreementwith previous studies.38 However, as x increases, so does the electronic contribution to PC, until γ reaches 29.6 mJ mol–1 K–2 for the x = 0.3 sample. Giventhese materials are Mott insulators, this electronic contributioncan be associated with only magnetic excitations, not conduction electrons.Zero-field heat capacity(46+ is site-selectively doped onto the corner-sharing B″(c) site.As illustrated in Jrung and Jleg, interactions are most affectedby W6+ doping of Ba2CuTeO6. The minoroccupation (<5%) of the B″(f) site is unlikely to have asignificant effect on the interladder interactions. Hence, d10/d0 doping on the B″(c) site directly tunes theintraladder Cu–O–B″(c)–O–Cu superexchange,while the interladder Cu–O–B″(f)–O–Cuexchange remains unchanged  and Te6+ (0.56 Å).55 The hexagonal layered structure and, hence,the Cu2+ spin ladder geometry remained intact across thesolid solution. There are only a few minor differences. Mainly, thevariable-temperature neutron diffraction data show W6+ dopingreduced the C2/m to P1 structural transition temperature from justbelow room temperature (x = 0) to ∼100–120K (x = 0.3). The C2/m to P1 transition is weak;therefore, the spin ladder structure and magnetic interactions remainthe same.W6+ to reside on the corner-sharing B″(c) site.Across the Ba2CuTex1–WxO6 series, the structuralmodel provided the best fit to the data when the majority (∼95%)of the W6+ dopant present in the sample resided on theB″(C) site. The EXAFS data corroborated this result, reproducingthe experimental data only when the model placed W6+ exclusivelyon the B″(c) site. The strong site selectivity appears to besurprising for a few reasons, first due to the aforementioned nearlyidentical W6+ and Te6+ ionic radii.56 This, and the identical +6 charge, lends usto expect a random distribution of Te6+ and W6+ across the B″(c) and B″(f) sites. However, this argumentneglects consideration of the metal–oxygen bonding differencesin Te6+ and W6+ (as well as Mo6+)perovskite structures.The synchrotron X-ray data revealed the strong selectivityforW6+ andMo6+ exclusively form double perovskite structures, whereasTe6+-containing perovskites can also adopt hexagonal structures.57 The W6+ and Mo6+ cationsinability to form hexagonal structures stems from the differencesin metal–oxygen bonding involving d0 versus d10 cations. In the case of Te6+, the filled 4d10 orbitals limit the d-orbital contribution to metal–oxygenbonding, creating a significant s- and p-orbital contribution in Te–Obonding. This directs the electron density toward the oxide anionsand away from the octahedral surface, thus helping to weaken cation–cationrepulsion between Te6+ and the surrounding B′ cation.57 The weakened cation–cation repulsionallows Te6+ to occupy face-sharing sites in hexagonal perovskitestructures, such as the B″(f) site in Ba2CuTex1–WxO6. The opposite is true for W6+ and Mo6+ perovskites where the 5d0 orbitals do contribute significantlyto metal–oxygen bonding, leading to a strong π-orbitalcontribution. These π-bonding interactions generate highly regular[WO6]6– octahedral units, with a morespherical charge distribution.58 This producesa relatively strong repulsion across shared octahedral faces, makingface-sharing unfavorable. Consequently, W6+ and Mo6+ prefer corner-sharing sites where cations are farther apartand therefore do not form hexagonal structures.It has been noted that perovskites containingW6+ strongly prefers thecorner-sharing B″(c) site, where thedistance to the Cu2+ cation is significantly larger thanthat of the face-sharing B″(f) site in the Cu–B″(f)–Cutrimer, e.g., Te6+–Cu2+ distances of3.962(2) Å (face-sharing) and 2.738(1) Å (face-sharing)in x = 0.3 at 300 K. The unfavorability of the B″(f)site also explains why attempts to synthesize richer W6+ compositions beyond x = 0.3 failed. Furthermore,this reasoning is also the basis for why Ba2CuTeO6 and Ba2CuWO6 adopt different structures. Thedifference in metal–oxygen bonding drives W6+ toform tetragonal Ba2CuWO6 to maximize the cationdistances, while Te6+ can accommodate face-sharing in hexagonalBa2CuTeO6.57 Notethat while seemingly stronger covalency in Te–O may imply strongersuperexchange interactions, this is not necessarily the case. Superexchangebetween corner-sharing CuO6 and TeO6 octahedramainly occurs via a Cu–O–O–Cu pathway withouta significant contribution from the Te6+ 4d10 states, which lie far below the Fermi level, or the Te6+ 5s and 5p states.13 In contrast, the empty W6+ 5d0 orbitals hybridize strongly with O 2p, resultingin significant Cu–O–W–O–Cu superexchange.This results in different dominant interactions for Te6+ and W6+ compounds, although the prediction of the overallstrength of magnetic interactions remains difficult due to competingeffects.This electrostaticenergetic penalty explains why W0 W6+ cations were site-selectivelydoped onto the B″(c) site, which connects the intraladder Jleg and Jrung interactionsvia Cu–O–B″(c)–O–Cu superexchange.Therefore, one would expect the d10/d0 dopingto result in the direct tuning of these intraladder interactions inBa2CuTex1–WxO6. This does in fact happen as confirmedby our isolated spin ladder fits to magnetic susceptibility data.Tungsten doping has a significant effect on the relative strengthsof the intraladder interactions: the Jrung/Jleg ratio decreased from ∼1for the x = 0 sample as the W6+ contentincreased, reaching a Jrung/Jleg value of ∼0.1 for the x =0.3 sample. Jleg was approximately constantwith an increase in x, therefore showing decreasesin Jrung/Jleg originate from a weakening of the Jrung interaction. Such a modification of the relative intraladder strengthby W6+ means that as Jrung/Jleg approaches zero, the system is progressivelytuned from a spin ladder toward an isolated spin chain.The d6+ doping has a significanteffect on the magnetic interactions in Ba2CuTex1–WxO6, thetrue magnetic ground states of the doped samples remain unknown. Weare unable to rule out the presence of magnetic order in the dopedsamples despite the lack of magnetic Bragg peaks in the neutron diffractionpatterns. This is because magnetic scattering from Cu2+S = 1/2 moments is very weakand HRPD is an instrument optimized for structure solution as opposedto magnetism.Whileour results show that W2CuTex1–WxO6 mightchange upon doping. We observe an increasing Curie tail in the dcsusceptibility with an increase in the level of d10/d0 doping. More importantly, a significant T-linear γ term is observed in the heat capacity data for dopedsamples, but not for pure Ba2CuTeO6. This largeγ term has no obvious origin in a magnetically ordered insulator.The high degree of Te/W disorder on the B″(c) site along withmagnetic frustration could lead to a spin glass state, which wouldexplain the γ term in the heat capacity.59 However, our ac susceptibility measurements show no evidenceof a spin glass state down to 2 K. Another possibility is that a quantumdisordered state, such as a random singlet state, might form in Ba2CuTex1–WxO6 as it forms in Sr2CuTex1–WxO6. Thiswould explain both the Curie-tail feature in the magnetic susceptibilityand the γ term in the heat capacity.61 For the x = 0.3 sample, the γ term approaches50% of the value for Sr2CuTe0.5W0.5O6.8 The ground state of thedoped samples could be further investigated using muon spin rotationand relaxation and additional neutron scattering experiments.Magnetic susceptibility and heat capacity dataprovide hints thatthe magnetic ground state of Ba52CuTeO6 delivers a Ba2CuTex1–WxO6 solid solution(0 ≤ x ≤ 0.3). Structural differencesamong the x = 0, 0.05, 0.1, 0.2, and 0.3 sampleswere investigated using a combination of neutron diffraction, synchrotronX-ray diffraction, and EXAFS. This revealed a strong site selectivityfor W6+ cations to occupy the corner-sharing B″(c)site within the intraladder structure. The site selectivity resultsfrom differences in molecular bonding that leads W6+ toprefer the corner-sharing site. Site-specific d10/d0 doping directly modifies the intraladder interactions bysuppressing Jrung as the level of W6+ doping increases, while Jleg remains constant. While it is unclear what type of ground statethis creates, it is clear the direct d10/d0 effecthas a significant effect on the magnetic interactions. As the levelof W6+ doping increases, Jrung is further suppressed, and the system is tuned from a spin laddertoward a spin chain as Jrung/Jleg approaches zero.Chemical doping of the hexagonal perovskiteBa10/d0 effect can be extended beyonddouble perovskite structures to modifythe magnetic interactions in hexagonal perovskites. Furthermore, theeffect could be extended to any structural type that contains corner-sharingoctahedra, such as perovskite-derived two-dimensional structures .This could even be done in a site-selective manner as we have shownhere for Ba2CuTex1–WxO6. Therefore, our work highlightsthe d10/d0 effect as a powerful tool for tuningmagnetic interactions and ground states in perovskite-derived oxides.Overall, this work demonstratesthat the d


Bombyx mori VgR (BmVgR) binds and transports lipoprotein-1 (BmLP1) and lipoprotein-7 (BmLP7) of the 30 kDa lipoproteins (30 K proteins), which are essential for egg formation and embryonic development in B. mori. Protein sequence analysis showed BmLP7, similar to reported lipoprotein-3 (BmLP3), contains the cell-penetrating peptides and Cysteine position, while BmLP1 has not. Assays using Spodoptera frugiperda ovary cells (sf9) indicated the direct entry of BmLP7 into the cells, whereas BmLP1 failed to enter. However, co-immunoprecipitation (Co-IP) assays indicated that BmVgR could bind BmLP1. Western blotting and immunofluorescence assays further revealed that over-expressed BmVgR could transport BmLP1 into sf9 cells. Co-IP assays showed that SE11C (comprising LBD1+EGF1+OTC domains of BmVgR) or SE22C (comprising LBD2+EGF2+OTC domains of BmVgR) could bind BmLP1. Over-expressed SE11C or SE22C could also transport BmLP1 into sf9 cells. Western blotting revealed that the ability of SE11C to transport BmLP1 might be stronger than that of SE22C. In the vit mutant with BmVgR gene mutation (vit/vit), SDS-PAGE and western blotting showed the content of BmLP1 in the ovary, like BmVg, was lower than that in the normal silkworm. When transgenic with hsp70 promoter over-expressed BmVgR in the vit mutant, we found that the phenotype of the vit mutant was partly rescued after heat treatment. And contents of BmLP1 and BmVg in vit mutant over-expressed BmVgR were higher than in the vit mutant. We conclude that BmVgR and its two repeat domains could bind and transport BmLP1 into the oocytes of the silkworm, besides BmVg. These results will provide a reference for studying the molecular mechanism of VgR transporting ligands in insects.Vitellogenin receptors (VgRs) transport vitellogenin (Vg) into oocytes, thereby promoting egg growth and embryonic development. VgRs recognize and transport multiple ligands in oviparous animals, but their role in insects is rarely reported. In this study, we investigated whether  In insects, vitellogenin receptors (VgRs) belong to the low-density lipoprotein receptor (LDLR) family, which is able to transport Vg into the oocytes and provide nutrition for eggs growth and embryonic development . VgR is vit mutant lacks vitellin (BmVn) and 30 K proteins, although their precursors are abundant in the pupal hemolymph  and homozygous lethal eggs  domain. This led to the retention of its ligand-binding ability but hampered its ligand dissociation under acidic conditions. Thus, BmVgR was unable to return to the cell membrane to transport other ligands. Eventually, this led to the lack of BmVn and 30 K proteins in the eggs of the vit mutant , serine/threonine-rich lipoproteins (BmLP25–36), and ENF peptide-binding proteins (BmLP37–46) . 30 K prvit mutant and transgenic vit mutant were used to identify whether BmVgR could transport BmLP1 into silkworm oocytes.In addition to BmVg, BmVgR might also transport BmLP1, one of the 30 K proteins without CPP. In this study, we downloaded the 30 K protein sequences and analyzed their structure. Co-IP assay was performed to reveal whether BmVgR could bind to BmLP1 and whether the two ligand-binding domains (LBRs) of BmVgR were involved in the transport of BmLP1. Cell incubation assays were performed to determine whether BmVgR could transport BmLP1 into insect cells by pHrodo-Red labeling. Studies using the vit mutant heterozygote vit/+ strain (vit oh/+ +♀ × vit oh/vit oh♂) used in this study was obtained from Kyushu University (Silkworm Base). The vit mutant vit/vit  and dazao (wild type) strains were maintained at Southwest University, China. The silkworm transgenic BmVgR + vit (BmVgR overexpressed in vit mutant) strain was generated in our laboratory by repeatedly mating the transgenic BmVgR strain with the vit mutant. The transgenic BmVgR strain was developed using transgenic BmVgR/BmVgR-positive individuals selected by injecting the transgenic plasmid piggyBac-[3×p3-EGFP-SV40]-[hsp70-BmVgR-SV40] into silkworm eggs. The Hsp70 promoter used in the construct was that of the Drosophila melanogaster heat-shock protein 70 gene. All larvae were reared on fresh mulberry leaves; pupae and moths were maintained at 25°C, 75% ± 5% relative humidity, and a photoperiod of 12 h light/12 h dark. All hemolymph samples were collected into ice-cooled tubes containing a few of phenylthiourea crystals and 1 mM phenyl methyl sulfonyl fluoride (PMSF). After haemocytes were removed by centrifugation at 6,000 g at 4°C for 20 min, and stored at −80°C. Ovaries were dissected from female pupae and moths, washed using physiological saline with diethyl pyrocarbonate, and stored at −80°C.The silkworm Spodoptera frugiperda ovary cells). The silkworm ovaries from each phase were homogenized using liquid nitrogen. Tissue and cellular total proteins were extracted using a kit as per the manufacturer’s instructions . NP40 lysate buffer (Beyotime) (containing 1 mM PMSF) was added to the powder or the cells, and the mixture was lysed on ice for 30 min. This was followed by centrifugation at 12,000 g for 10 min, and the resultant supernatant was collected.Protein samples were obtained from the ovaries at different phases in the silkworm pupae or from sf9 cells  under a ChemiScope Imaging System.The supernatant was subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis  as described by Laemmli . The geldazao hemolymph as previously described  at 4°C. The dialyzed sample was then purified using Q column (HiTrapQ HP anion exchange column). After verification using SDS-PAGE, the desired proteins were gathered and applied to a HiLoad Superdex S-75 16/600 column purification . The desired proteins were collected. The purified proteins were further confirmed using SDS-PAGE. The recombinant BmLP1 protein (His-BmLP1) was generated in our laboratory (The BmLP1 and BmLP7 proteins were purified from silkworm escribed . On day boratory .The 1180 (Hrs1000-BmActin4-SV40) expression vector was maintained in our laboratory. We obtained full-length BmVgR from p50 T6kb-9 plasmid DNA , 1180-BmVgR (Hrs1000-BmActin4-SP-LBD1-EGF1-LBD2-EGF2-OTC-Ser1PA), 1180-S11C (Hrs1000-BmActin4-SP-LBD1-EGF1-OTC-Ser1PA), and 1180-SP-EGFP-C (Hrs1000-BmActin4-SP-EGFP-C-Ser1PA) (stored at our laboratory).via homologous recombination PCR; SP-EGFP’ and LBD2-EGF2-OTC were used as templates. The PCR conditions were 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 2 min 40 s, and a final extension at 72°C for 10 min. All PCR products were confirmed by electrophoresis on a 1.2% (w/v) agarose gel and photographed using the Molecular Imager Gel Doc XR system .Before the SP-EGFP-LBD1-EGF1-OTC (SE11C) was obtained, we performed polymerase chain reaction (PCR) amplification of SP-EGFP and LBD1-EGF1-OTC. The primers used for PCR are listed in After the amplified fragments were confirmed by sequencing, the fragments were ligated into the 1180 expression vector using the Ligation-free Cloning System . The vectors were named SE11C and SE22C and were confirmed by sequencing. The sf9 cell line was used for cellular assays since the endogenous genes of BmVgR, BmLP1, and BmLP7 are not expressed in sf9 cells. The cells were transfected with SE11C or SE22C plasmid DNA using Cellfection II kit , following the manufacturer’s instructions. After the complete medium was replaced with fresh medium and the cells were cultured for 72 h, total protein from the cells was extracted using NP40 lysis buffer (Beyotime); the expression of SE11C or SE22C protein was analyzed using western blotting with anti-GFP polyclonal rabbit antibody   and HRP-conjugated goat anti-rabbit IgG (H + L) antibody  (Beyotime). The transfected cells were also used to determine the localization of existing SE11C or SE22C using a fluorescence assay. Detection of SE11C and SE22C in sf9 cells using green fluorescence under a laser confocal microscope .dazao on day one of pupation using western blotting with anti-LBD2 polyclonal rabbit antibody . Recombinant antigen LBD2 (amino acid positions of BmVgR 934–1282) was expressed using the pET28a-BmVgR-LBD2 plasmid in Escherichia coli strain BL21 (DE3) . SE11C/SE22C protein was obtained by expressing SE11C/SE22C in sf9 cells and detected using western blotting with anti-GFP polyclonal rabbit antibody . His-BmLP1 protein was added to the culture medium and incubated with the BmVgR/SE11C/SE22C protein at 4°C for 6 h. Approximately 7.5 μg of anti-His mouse monoclonal antibody  was then diluted in 200 μL of NP40 and added to 50 μL of 5% (w/v) BSA-blocked Dynabeads (Thermo Fisher Scientific). The Dynabeads were used for the experiment according to manufacturer’s instructions. The incubation medium was collected as a positive control. Interference of non-specific proteins was excluded by washing the membrane twice with phosphate-buffered saline  and then once with PBS (pH 7.4), and the final wash buffer was used as a negative control. The complex was resuspended in 100 μL of PBS (pH 7.4) and transferred to a clean tube. Approximately 20 μL of 5 × SDS–PAGE loading buffer was then added, and the mixture was heated for 10 min at 100 °C. The eluted protein from the beads was subjected to SDS–PAGE (10% w/v polyacrylamide gel) and detected using western blotting with anti-LBD1 against recombinant antigen LBD1 (amino acid positions of BmVgR 27–224) expressed using pET28a-BmVgR-LBD1 in Escherichia coli strain BL21 (DE3)  or anti-LBD2 rabbit antibody .BmVgR protein was detected in the total protein samples of the ovaries of the silkworm 3 buffer (pH 8.3), and the proteins were diluted to 1 mg/mL prior to fluorescent labeling. An appropriate amount of reactive dye (dye 10: protein 1) was added to the protein solution in sodium bicarbonate buffer and mixed. The reaction was incubated for 1 h at 25 °C in the dark. The labeled BmLP1 and BmLP7 proteins were purified using ultrafiltration in the dark. The pHrodo-Red-labeled BmLP1 and BmLP7 proteins (BmLP1-Red and BmLP7-Red) prepared were stored at −80 °C until further use.Fluorescent labeling of BmLP1 and BmLP7 proteins was performed using pHrodo-Red dye (Thermo Fisher Scientific), following the manufacturer’s instructions. The stock solution of the dye was prepared by dissolving 1 mg pHrodo-Red dye into 150 μl of dimethyl sulfoxide. The buffer for BmLP1 and BmLP7 proteins were replaced with freshly prepared 0.1 M NaHCOThe mechanism of entry of BmLP1 into sf9 cells was studied using 12-well cell culture plates (for western blotting) and 24-well cell culture plates (for immunofluorescence); BmLP7 was used as the control. Sf9 cells were starved for 30 min in Grace’s medium without fetal bovine serum and incubated with 200 μg/mL BmLP1-Red or BmLP7-Red protein for 60 min at 27°C. The cells were washed twice with PBS (pH 6.5) and then once with PBS (pH 7.4) to remove ligand proteins that failed to enter the cells. BmLP1 and BmLP7 proteins were detected using western blotting analysis with anti-BmLP1 and anti-BmLP7 rabbit antibodies  (anti-BmLP1 and anti-BmLP7 rabbit antibodies were prepared in our laboratory) , respectTo determine whether BmLP1 is transported by BmVgR, SE11C, or SE22C, each of these three proteins was overexpressed in sf9 cells and then analyzed using the methods described above. BmVgR and BmLP1 proteins were detected using western blotting with anti-LBD2 rabbit antibody  or anti-BmLP1 rabbit antibody , followed by HRP-conjugated goat anti-rabbit IgG (H + L) antibody . And the cells detected using immunofluorescence assay with anti-LBD2 rabbit antibody and goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody , Alexa Fluor™ 488 (Thermo Fisher Scientific); anti-BmLP1 rabbit antibody  and goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody , Alexa Fluor™ 594 (Thermo Fisher Scientific).vit mutant, vit/+, dazao, and BmVgR + vit strains during the moth stage; the samples were flash-frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted using the TRIzol Reagent kit ; Moloney Murine Leukemia Virus Reverse Transcriptase  was used to generate first-strand cDNA. All assays were done according to manufacturers’ instructions.Ovaries were dissected from dazao, the vit mutant had abnormal BmVgR, with a 228-bp deletion in the third-class B region of the first EGF domain (vit-deleted gene fragment (abbreviated as vit’). The forward primer (vit’-F) was 5ʹ-CTCCATATGTACCACCCAGCGTTGA-3ʹ, and the reverse primer (vit’-R) was 5ʹ-GGGGATGCATCTGCCGTTCTTGTTT-3ʹ. The PCR conditions were 94°C for 5 min, followed by 27 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 15 s, and a final extension at 72°C for 10 min Bmactin3 was used as an internal reference; the forward primer was 5ʹ-AACACCCCGTCCTGCTCACTG-3ʹ, and the reverse primer was 5ʹ-GGGCGAGACGTGTGATTTCCT-3ʹ. PCR was performed at 94°C for 5 min, followed by 25 cycles of 94°C for 30 s, 53°C for 30 s, and 72 °C for 30 s, and a final extension at 72°C for 10 min. The vit’ and Bmactin3 cDNA fragments were PCR-amplified using rTaq DNA polymerase . The PCR products were confirmed by electrophoresis on 1.2% (w/v) agarose gel and photographed using the Molecular Imager Gel Doc XR system .Unlike F domain . PrimersBmLP1, BmLP3, and BmLP7 belong to the 30 K family. We compared the sequence of CPP-BmLP3 with 46 types of 30 K proteins in the silkworms. The results indicated that BmLP7, BmLP8, BmLP9, BmLP10, BmLP14, besides BmLP3 (amino acid residues marked with red boxes) contained the CPP domain and Cysteine position . HoweverTo analyze whether BmLP1 (without CPP domain and Cysteine position) and BmLP7 (with CPP domain and Cysteine position) could enter into the sf9 cells without BmVgR, we used pHrodo-Red dye to label the BmLP1 and BmLP7 proteins, which were purified from the hemolymph at day two of silkworm pupation. SDS-PAGE indicated rose-red bands under white light, which represented BmLP1-Red  and BmLPin vitro assay to determine the interaction between BmVgR and BmLP1. Western blotting analysis revealed the presence of BmVgR in the ovaries of the silkworm dazao on day one of pupation  and the LBD2+EGF2+OTC domain (SE22C) of BmVgR could transport BmLP1, we constructed truncated cell expression vectors with a SE11C or SE22C structure . FluoresTo confirm the binding of SE11C and SE22C with BmLP1, His-BmLP1 was incubated with the lysate of sf9 cells with SE11C and SE22C over-expression, respectively. Co-IP showed that both SE11C  and SE22Further, we investigated whether SE11C and SE22C could transport BmLP1 into sf9 cells. We incubated SE11C or SE22C over-expressed sf9 cells with 1 mg/mL BmLP1. We performed western blotting to detect BmLP1 in sf9 cell lysates. The results showed that BmLP1 was transported into the sf9 cells over-expressing SE11C or SE22C . Throughvit mutant was due to BmVgR not normally transporting ligands into oocytes, as exhibited by BmVg and 30 K proteins ; vit/+ was used as a control. Twenty female pupae from each strain were treated at 27°C, 32°C, 37°C, and 42°C for 1 h on 1, 3, 5, and 7 days of pupation, respectively. Control strains were treated at 27 °C from corresponding stages of pupation with sample strains. After the heat shock treatment, we checked the presence of deleted fragments of the vit mutant (vit’) in the ovaries of the heat shock-treated and control strains by RT-PCR. The results showed that vit’ was detected in the ovaries of BmVgR + vit, as well as those of the control post heat shock-treatment. However, the vit’ of the BmVgR + vit was not detected like that of the vit mutant with no heat shock treatment  and BmLP7 (with CPP domain and Cysteine position) for our experiments. Our results indicated that 30 K proteins with CPP domain and Cysteine position, such as BmLP3/BmLP7, could gain direct entry into the cells independent of the receptors. The transportation of 30 K proteins, such as BmLP1, which do not contain the CPP domain and Cysteine position, might be mediated by receptors.Like BmVn, 30 K proteins are important components of egg yolk proteins in silkworm eggs, providing nutrients for egg formation and later embryonic development. Park et al. discovered that BmLP3, one of the 30 K proteins with a CPP and Cysteine position, could efficiently penetrate cells grown in a medium used for mammalian cell culture . HoweverCo-IP of total proteins from the ovaries incubated with the His-BmLP1 obtained from prokaryotic expression showed that BmVgR could bind with His-BmLP1. BmLP1-Red, when incubated with sf9 cells over-expressing BmVgR, showed that BmLP1-Red proteins could be transported into the sf9 cells over-expressing BmVgR. We found that the additional LBD1+EGF1+OTC (SE11C) and LBD2+EGF2+OTC (SE22C) domains could also transport BmLP1 into the cells; the ability of SE11C to transport BmLP1 might be stronger than that of SE22C. The results obtained using western blotting, Co-IP, and fluorescence imaging revealed that BmVgR with different LBDs could transport multiple ligands. These results correspond to those of previous studies which demonstrated that VgRs recognize multiple ligands . LRP recBmVgR mutant as the experimental material for individual biological verification. The vit mutant represents the abnormal function of BmVgR owing to the lack of BmVn and 30 K proteins in the ovary  that multiple forms of VtgR bind to multiple types of Vtg (Oreochromis aureus, Lys185, a positively charged residue, plays a crucial role in the receptor binding of Vtg (We used the he ovary . We useds of Vtg . The LBDg of Vtg . Yet, thvia receptor-independent endocytosis (CPPs and their protein transduction domains have been used to deliver drugs and proteins into the cells ocytosis . BmLP3/Bocytosis . In the ocytosis . Since s


Under the condition for cellulase production,disruption of gul1 resulted in smaller mycelial clumps andsignificantly lower viscosity of fermentation broth. In addition, cellulaseproduction was improved by 22% relative to the parent strain.Transcriptome analysis revealed that a set of genes encoding cell wallremodeling enzymes as well as hydrophobins were differentially expressed in theΔgul1 strain. The results suggest that theregulatory role of gul1 in cell morphogenesis is likelyconserved in filamentous fungi. To our knowledge, this is the first report onthe engineering of gul1 in an industrially importantfungus.Hyphal morphology is considered to have a close relationship with the productionlevel of secreted proteins by filamentous fungi. In this study, the Trichoderma reesei (teleomorphHypocrea jecorina) is widely used for industrial cellulaseproduction in the world  kinaseCOT-1 comprise a pathway regulating cell wall integrity and morphogenesis inN. crassa  is regulated by CBK1 (COT-1 homolog) through phosphorylation  plates at 30°Cfor 7 days for conidiation. The conidia were harvested by washing PDA plateswith distilled water containing 0.9% (wt/vol) NaCl and 0.01%(wt/vol) Tween 80. For mycelial growth study, fresh conidia were inoculated into50 ml minimal medium at a final concentration of 104)2SO4 5.0, KH2PO415.0, MgSO4·7H2O 0.6, CaCl2 0.6, peptone2.0, FeSO4·7H2O 0.005,MnSO4·H2O 0.0016,ZnSO4·7H2O 0.0014, andCoCl·6H2O 0.002. The cellulase production medium contained(g/l): microcrystalline cellulose 20.0, corn steep liquor 20.0,KH2PO4 5.0, (NH4)2SO42.0, MgSO4·7H2O 0.6, and CaCl2 1.0.The minimal medium contained (g/l): glucose 20.0,(NH6 per ml) of strains wasinoculated on the center of PDA or cellulose agar plates, and then cultivated at30°C. The cellulose agar plate was the same with minimal medium agarplate except that glucose was replaced by 2% (wt/vol) ball-milledcellulose. The diameters of colonies on agar plates were measured every day. Forstress sensitivity analysis, the strains were inoculated on minimal mediumplates supplied with different chemicals as indicated, and cultivated at30°C unless specifically stated.One microliter of conidial suspension  value wascalculated by dividing the hyphal length by the number of tips ,and analyzed using the ImageJ 1.8.0 software NaCl solution. Next, the precipitate was resuspended in 1 ml of1 M NaOH solution and incubated at 200 rpm for 1 h at roomtemperature. The suspension was centrifuged at 8,000 gfor 10 min, and the protein content of the supernatant was determined bythe Bradford Protein Assay Kit .The mycelial biomass in 50 ml liquid minimal medium was collected byvacuum filtration and washed with distilled water. The mycelia were dried toconstant weight at 60°C and weighed. Due to the insolubility ofmicrocrystalline cellulose, the biomass in cellulase production medium wasmeasured indirectly by determining the amount of internal protein. Specifically,1 ml of culture broth was centrifuged at 8,000 The viscosity of fermentation broth was measured using digital viscometer NDJ-5S according to themanufacturer's instructions. The instrument drives a spindle (immersed inthe test sample) through a calibrated spring, and measures the viscous drag ofthe fluid against the spindle by the spring deflection. One hundred ml ofculture broth in a 100 ml beaker was used for measurement with threerepeats at room temperature. No. 2 spindle supplied with the viscometer was usedwith the rotation speed set at 6 rpm. Data in the measurement range of20– 40% were recorded after the readings were stable.g, 4°Cfor 10 min to collect supernatant. Filter paper activity was determinedwith Whatman No. 1 filter paper as the substrate as previously described  using HISAT2 .The strains were first grown in minimal medium for 36 hr. Then,5 ml culture was inoculated to 50 ml cellulase production mediumwith 10 g/l microcrystalline cellulose as the sole carbon source, andthen cultured for 48 hr in biological triplicates. Mycelia were harvestedby vacuum filtration and frozen immediately in liquid nitrogen. Total RNA wasisolated from ground mycelia using RNAiso Reagent  according tothe manufacturer's instructions. High-throughput sequencing of RNAsamples was performed by Personal Biotechnology Co., Ltd. .Briefly, mRNA was purified from total RNA using poly-T oligo attached magneticbeads, and then sequencing libraries were generated using the TruSeq RNA SamplePreparation Kit . The products with an averageinsert size of 380 bp were purified using the AMPure XP system , and quantified using the Agilent high sensitivityDNA assay on a Bioanalyzer 2100 system (Agilent). Paired-end sequencing wasperformed on a NovaSeq 6000 platform (Illumina) with a read length of150 bp. The clean reads obtained after raw data processing were mapped tothe reference genome of https://mycocosm.jgi.doe.gov/Trire2/Trire2.home.html). Theproteins were then used for the prediction of GPI anchored proteins using theonline tool NetGPI-1.0 .Statistical significance tests of differences were performed by calculatingT. reeseiwild-type strain QM6a as the ortholog of N. crassa GUL-1 is correct.Reciprocal BLASTp analysis identified the protein product of gene Trire2_77084(NCBI RefSeq accession number: XP_006964501.1) in T. reesei GUL1 has anidentity of 76.8% with N. crassa GUL-1. Althoughpredicted to have a ribonuclease II/R domain, T. reesei GUL1should not have a ribonuclease activity due to the lack of some key amino acidresidues  for catalysis, which is a commonfeature for GUL-1/Ssd1 orthologs .The 1,349 amino acid-long sequence of rthologs . In addigul1 gene was deleted via homologousrecombination in T. reesei QP4, a uridine auxotrophic strainconstructed from strain QM9414 . In this study, there is actually no significant difference betweenQP4 and QPP when uracil was supplemented in the culture medium.The major part of n Pronk, , QP4 wasgul1 strain showed a slower radialgrowth than reference strains QP4 and QPP  was lower thanthat of QPP (0.19 h−1). This result was consistent with thehigher germination rate of the conidia of Δgul1.According to microscopic observation, the mean percent of germination forconidia of Δgul1 was 24.84% (54/213) after10 hr of incubation, while that of QP4 and QPP was 14.00% (35/250)and 13.81% (25/181), respectively.The growth of strains was also compared in liquid medium with 2% (wt/vol)glucose as the sole carbon source. The Δion Fig. .Neverthgul1 strain showed a hyper lateral branchingphenotype on cellulose agar plate when examined with microscope clearly suggested thatgul1 disruption increased hyphal branching cellulose served as acarbon source. Under this condition, the strains formed more compact mycelialclumps relative to those in glucose medium. Similarly, smaller mycelial clumpswere observed for Δgul1 than the reference strains.Mycelial autolysis was observed after 120 hr, when the broth viscositydropped below 100 mPa s for both strains.Considering the wide use of insFig. . In addiionFig. . The visgul1 strain was higher than those of referencestrains  of theΔins Fig. . The celkDa Fig.  and c. Tgul1 was due to any change in cell biomassabundance, intracellular proteins were extracted and determined to indirectlystudy cell growth in cellulose medium  were compared using the RNA-seq technology.While Ssd1 is believed to mainly regulate the translation of its target mRNAs, itcould also stabilize at least some of the targets and therefore affect theabundance of transcripts , of which 14 were differentially expressed in inΔgul1 relative to QPP. These genes are predicted toencode a β-1,3-glucanosyltransferase, an α-1,6-mannanase, andputative cell wall mannoproteins with unknown functions  anchored proteins, which are known to beattached to membrane or cell wall in fungi, are involved in cell wallbiogenesis, integrity and cell adhesion  compared with the reference strain,which could explain its lower broth viscosity throughout fermentation.The viscosity of fermentation broths during cellulase production showed rapiddecreases between 24 and 48 hr, when cell biomass was still increasingFigs . This ingul1 strain are similarwith those of gul-1 deletion mutant in N. crassaand TRIREDRAFT_66792  were both downregulatedin Δgul1, and similar downregulations of their orthologswere also detected in N. crassa Δgul-1. Theorthologs of Aspergillus nidulans phiA, which is required fornormal phialide development, showed significantly increased transcript abundances inboth Δgul1 and Δgul-1. The changesin the transcript abundance of these genes might contribute to the alteredmorphologies of gul1/gul-1 disruption mutants.Many of the phenotypical changes observed in the Δin Δgul1, of whicgul-1 in N. crassa increased theproduction of extracellular β-glucosidase but not that of major cellulases has an ortholog in T. reesei(chi18-17). Therefore, Ssd1/GUL1 might evolve with differenttargets in different fungal species. The identification of GUL1-bound targets  is expected to reveal the regulatory mechanismof GUL1, which is important for the understanding and precise engineering of cellmorphogenesis in filamentous fungi.The Cbk1-Ssd1 pathway is similar between kuab012_Supplemental_FilesClick here for additional data file.


Amycolatopsis sp. DEM30355 has allowed for the discovery and isolation of the polyketide antibiotic tatiomicin. Identification of the biosynthetic gene cluster was confirmed by heterologous expression in Streptomyces coelicolor M1152. Structural elucidation, including absolute stereochemical assignment, was performed using complementary crystallographic, spectroscopic and computational methods. Tatiomicin shows antibiotic activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Cytological profiling experiments suggest a putative antibiotic mode-of-action, involving membrane depolarisation and chromosomal decondensation of the target bacteria.A genomic and bioactivity informed analysis of the metabolome of the extremophile  Actinobacteria are a rich source of antibiotic natural products, with developments in this area being fuelled by the application of next-generation whole-genome sequencing technologies in combination with advanced bioinformatic analysis14. The combination of modern genomic techniques with the more classical approach of focussing on understudied, extremophilic, “unculturable” (so far not cultured) bacteria or rare bacterial genera, provides a rich ground for the discovery of new mode-of-action antibiotics. We therefore decided to investigate the metabolome of the extremophile Amycolatopsis sp. DEM3035516, which was isolated from a soil sample collected from the El Tatio geyser field within an arid part of the Atacama Desert in Chile17, through a combined genomic and bioactivity informed isolation approach resulting in the discovery of a novel anthracenone polyketide, tatiomicin. Following structural assignment, absolute stereochemistry was determined through a combination of crystallographic (resonant scattering), spectroscopic and computational methods, supporting the reassignment of the absolute stereochemistry of its nearest known natural product congeners the rishirilides18. Tatiomicin shows selective antibiotic activity against a focussed group of Gram-positive bacteria, including clinically relevant MRSA. Mode-of-action experiments indicated that tatiomicin may act as a Michael acceptor in vitro, an electrophile that can undergo conjugate addition reactions with biomolecules, and exhibits its antibiotic activity via a pathway involving membrane depolarisation and chromosomal decondensation.Treatment of resistant infectious diseases poses a significant threat to human health, in particular those arising from the emergence of multidrug-resistant pathogenic bacteria. This has prompted significant efforts in the development of new small molecule antibiotics17. Strain DEM30355 was recovered in the genus Amycolatopsis, based on 16S rRNA analysis, forming a subgroup with Amycolatopsis vancoresmycina DSM 44592 T and Amycolatopsis bullii SF27T (see ESI). The genus Amycolatopsis contains 94 species and four subspecies encompassing both extremophiles and producers of bioactive secondary metabolites, including the clinically used vancomycin and rifamycin antibiotics20. Preliminary bioactivity screening showed that extracts of Amycolatopsis sp. DEM30355 displayed promising antibiotic activity against B. subtilis, thus we decided to examine the genome of Amycolatopsis sp. DEM30355 for novel biosynthetic potential. Purified genomic DNA from Amycolatopsis sp. DEM30355 was analysed using both PacBio® and Illumina® sequencing technologies and genome assembly was performed using the combined datasets to give a 9.6 Mb draft genome, in 13 contigs. The draft genome of Amycolatopsis sp. DEM30355 was examined using the secondary metabolite analysis software AntiSMASH 6.0.121. Of the 31 biosynthetic gene clusters (BGCs) detected, a PKS cluster was identified showing moderate overall similarity (81%) to that which encodes for rishirilides A and B26. These compounds are anthracenone polyketides, originally isolated from Streptomyces rishiriensis OFR-1056, with no reported antibiotic activity. Rishirilide B has been shown to be a moderately potent inhibitor of α2-macroglobulin, glutathione S-transferase and asparaginyl-tRNA synthetase, whilst little is known about the biological role of rishirilide A , compared to the rishirilide BGC, along with the presence of several new genes: one postulated to be involved in PKS biosynthesis (tatS1), two encoding methyltransferases (tatM1 and tatM2), two encoding cyclases (tatC4 and tatC5) and one cytochrome p450 oxidoreductase (tatO11) -tatiomicin (3)). Fermentation of Amycolatopsis sp. DEM30355, removal of the biomass, extraction of the supernatant and bioactivity guided fractionation by multiple chromatography steps resulted in a fraction which retained antimicrobial activity and contained two closely related compounds. HRMS analysis suggested that these compounds were stereoisomers of each other, the major compound showing m/z = 425.1221 [M + Na]+ corresponding to a molecular formula of C21H22O8 for both molecules (see ESI).Preliminary analysis of the fermentation supernatant of 3)  analysis, revealing a highly oxygenated anthracenone polyketide, structurally consistent with the BGC of interest, which we named (–)-tatiomicin (3) Fig. 29.C-2 epimer, capable of equilibrating with (–)-(3) under acidic conditions (see ESI).NMR and HPLC experiments demonstrated that the minor compound was the Determination of the absolute stereochemistry of (–)-3 was undertaken in parallel via vibrational and electronic circular dichroism spectroscopies and additional single-crystal X-ray diffraction (SCXRD) experiments.IR = 92.0, ΣVCD = 71.2, ESI = − 57.8) suggesting an absolute stereochemical assignment of   experiments; however, in this case correlation between experiment and prediction was weaker (see ESI).Absolute configuration determination by vibrational circular dichroism (VCD) was based on a comparison of experimental and computationally predicted spectra, taking into account the presence of two epimers of (–)-3. Conformational analysis (see ESI), removal of redundant geometries and final optimization at the B3LYP/6–311 +  + G level allowed Boltzmann-weighted VCD spectra for both epimers of (–)-3 to be constructed. The final predicted spectrum was obtained by applying a 3:1 ratio to account for the experimentally analysed mixture of epimers. Numerical analysis was used to establish agreement between experiment and theory, the neighbourhood similarity values (Σ3) was grown via slow evaporation from a benzene solution. Due to the crystal’s dimensions, diffraction data were collected at beam line I19 at the Diamond Light Source using synchrotron radiation at standard operating wavelength (λ = 0.6889 Å), providing a data set of sufficient quality to allow for structural confirmation. (–)-Tatiomicin (3) crystallized as an H-bonded dimer in the unit cell (Z’ = 2) along with a single molecule of solvent (benzene). To validate the absolute stereochemical assignment a further single-crystal X-ray diffraction experiment was undertaken at I19, employing non-typical, longer wavelength synchrotron radiation (λ = 1.4879 Å) to enhance resonant scattering contributions . The absolute-structure (‘Flack’) parameter (0.05(6)) was insignificantly different from zero and with a small standard uncertainty, indicating the correct absolute configuration in the refined  structure (see ESI)29. Interestingly, following extensive stereochemical debate and several reported total syntheses, the absolute stereochemistry of the congeneric (+)-rishirilide B (2) was recently revised , matching that of (–)-(3) over the three common stereocentres, suggesting a similar biosynthetic pathway for both sets of natural products , a high molecular-weight P1 artificial chromosome (PAC) library was obtained, consisting of 2,688 clones with an average insert size of 138 kb which contained resistant markers for kanamycin (for E. coli) and thiostreptone (for S. coelicolor). The PAC library was screened by PCR, using four primer pairs for the putative BGC. A single PAC clone was identified with the required PCR profile, which was then transferred into E. coli strain ET12567/pR9604 (dam- dcm-), the plasmid was subsequently transferred into S. coelicolor M1152 via conjugation. Exconjugants containing the plasmid integrated on the chromosome were selected for resistance to thiostrepton. Ninety-six putatively identified exconjugants were arrayed into 24 well plates and screened for the production of tatiomicin (3) by TLC, with detection based on the characteristic fluorescence upon UV irradiation at 365 nm. Based on these screening parameters, S. coelicolor M1152::tat was identified as a producer of tatiomicin (3) (see ESI).To verify that the BGC previously identified does indeed encode the biosynthetic pathway for tatiomicin  and analysed by LCMS alongside similar fermentation extracts from both the parent strain M1152, Amycolatopsis sp. DEM30355 and a tatiomicin (3) standard. An LCMS peak corresponding to tatiomicin (3) was observed in the extract from S. coelicolor M1152::tat but was absent in that of the parent strain M1152  was subsequently isolated from the fermentation of S. coelicolor M1152::tat in liquid medium (GYMG), as demonstrated by HRMS ([M + H]+ = 403.1403), with a production level in the heterologous host estimated at 0.57 mg/L, confirming the identity of the tatiomicin BGC  (See ESI). The modular type I polyketide synthase TatS1 is likely responsible for the biosynthesis of the polyketide starter unit, cis-crotonyl-ACP, which is then elongated via the attachment of eight malonyl-CoA by minimal PKS enzymes TatK1, TatK2, and TatK3. TatC1, TatC2, TatC3 and TatO10 show close homology to rishirilide cyclases RslC1, RslC2, and RslC3 and C9-ketoreductase RslO10, respectively. Thus, TatC1 and TatO10 likely act together to form the A ring of tatiomicin (3), whilst TatC2 and TatC3 catalyse the formation of the B and C rings. Tailoring of the polyketide core likely involves oxidation of the C ring by TatO4, and installation of the C ring epoxide by flavin mononucleotide (FMN)-dependent monooxygenase TatO1 together with a putative flavin reductase, TatO2. Opening of the epoxide is proposed to be mediated by NADPH:acceptor oxidoreductase TatO5, followed by the key Baeyer–Villiger oxidation/rearrangement controlled by TatO9 and finally reduction of the B ring ketone by ketoreductase TatO8.Based on a comparison between the tatiomicin and rishirilide BGCs3) for which no homologues are present in that of rishirilide, TatO11, TatM1 and TatM2. TatO11 is a cytochrome p450 oxidoreductase, likely responsible for oxidation of the A ring to the hydroquinone form, followed by double methylation by the two methyl transferases TatM1 and TatM2 to yield the completed molecule  showed no detectable antimicrobial activity (MIC > 64 µg/mL) against ten Gram-negative bacteria and two eukaryotic microorganisms (Candida spp.) (see ESI). However, antibacterial activity was observed against a sub-set of Gram-positive bacteria (MIC = 4–8 µg/mL), namely Staphylococcus and Streptococcus species. Due to the interest in developing new antibiotics against drug-resistant Staphylococcus infections, we further evaluated (–)-3 against a panel of MRSA clinical isolates, including twenty-four EMRSA-15 and EMRSA-16 strains 36, and twelve MRSA strains isolated from Belgian, Finnish, French and German hospitals (see SI). In all cases antibiotic activity was maintained (MIC = 4–8 µg/mL), suggesting that (–)-3 does not operate via a mode-of-action previously encountered by these strains, prompting us towards further investigation.(–)-Tatiomicin (Bacillus subtilis mutants resistant to (–)-tatiomicin (3) proved unsuccessful (see ESI). Also, no positive responses were seen with a panel of B. subtilis strains containing lacZ reporter genes used to indicate common antibacterial mechanisms of action, including: fatty acid synthesis (fabHA), DNA damage (ɸ105 prophage induction), RNA polymerase (RNAP) inhibition (helD), cell wall damage (ypuA), gyrase inhibition (gyrA), and cell envelope stress (liaI)) (see ESI)39.Elucidation of the mode-of-action (MOA) for a new antibacterial agent is a significant experimental challenge. The characterization of resistance mutations can be informative, however all attempts to isolate 3) may involve the covalent modification of thiol-containing enzymes through a conjugate or Michael addition of the thiol to the α,β-unsaturated carbonyl. Thus, (–)-tatiomicin (3) was reacted with L-cysteine hydrochloride, L-cysteine methyl ester hydrochloride and a short thiol-containing peptide (LcrV (271–291)) as an enzyme proxy, under biologically relevant conditions. In all cases thiol adducts could be detected by LCMS, suggesting that (–)-tatiomicin (3) may have biologically relevant Michael acceptor activity (see ESI).Due to the presence of an, albeit electron-rich, α,β-unsaturated carbonyl moiety, we postulated that the observed biological activity of (–)-tatiomicin -tatiomicin (3) induced chromosome decondensation in B. subtilis 168CA-CRW419, similar to the effects elicited by the RNAP inhibitor rifampicin  is used in parallel with Sytox Green, a membrane-impermeable DNA stain used as a reporter for pore formation44. Upon addition of nisin, which forms large pores in the B. subtilis membrane42, both a loss of DiSC3(5) and uptake of Sytox Green was observed. In contrast gramicidin, which forms small cation-specific channels45, showed loss of DiSC3(5) without Sytox Green staining. Treatment with (–)-tatiomicin (3) showed a similar effect to that of gramicidin, i.e. loss of DiSC3(5) without Sytox Green staining. Hence tatiomicin probably acts to dissipate the membrane potential without the formation of large pores . This dye accumulates in well-energised cells in the cytoplasmic membraneres Fig. .Figure 8In an attempt to ascertain whether the observed loss of membrane potential is a downstream effect or occurs at the same time as chromosome depolarisation we performed a time-course experiment using DiSC3(5) in combination with a HsbU-GFP fusion to assess chromosome decondensation with images taken every two minutes. This showed that the loss of membrane potential occurred simultaneously with the chromosome decondensation, between 2 to 4 min, suggesting that they are closely linked events Fig. .Figure 93) from Amycolatopsis sp. DEM30355, isolated from an hyper-arid Atacama desert soil, which shows antibiotic activity against a range of Gram-positive bacteria including MRSA clinical isolates. Comprehensive structural elucidation included absolute stereochemical assignment by complementary VCD and single crystal X-ray diffraction analysis, using long wavelength synchrotron radiation. The biosynthetic gene cluster of (–)-tatiomicin (3) was identified and validated through expression in a heterologous host, allowing us to propose a biosynthetic pathway. Single-cell microscopy studies suggest a pleiotropic mode-of-action involving membrane depolarisation and chromosomal decondensation, potentially involving the covalent modification of a target protein(s), although a definitive mode-of-action remains elusive at this time. Thus, we have demonstrated that modern bioprospecting techniques when applied to actinobacteria from extreme environments can still result in the discovery of new natural products with important bioactivity.We have identified (–)-tatiomicin  into two conical flasks filled with 10 mL of GYMG media . After 2 days of growth, cells were transferred into a 200 mL wv (working volume) flask. After incubation overnight, the two flasks were combined and an 8 L wv bioreactor was inoculated. An overnight incubation of a 50 L seed bioreactor was inoculated with 2.5 L of culture broth. Incubation for 15 h resulted in 40 L of exponentially growing cells at an OD450nm of 5.4. The 500 L wv bioreactor was inoculated with 25 L of this culture broth. The temperature was controlled at 30 °C. The dissolved oxygen was controlled at 50% via the agitation speed (starting speed 400 rpm). Compressed air was applied to the vessel at 1 vvm. The pH was adjusted to pH 7 prior to autoclaving.Biomass was removed via centrifugation  and filtration Satorious depth filters). Amberlite XAD-16 absorption resin (10 kg) was applied to the supernatant. After overnight incubation under agitation, the beads were filtered off and washed with deionised water. Elution using methanol (40 L) was carried out. The solvent was evaporated to an aqueous extract. The pH of the aqueous extract was adjusted to 4 using sulphuric acid and twice extracted with ethyl acetate. The organic layer was evaporated to dryness resulting in 231.11 g of crude extract.An aliquot (~ 4 g) of ethyl acetate extract, obtained from a 500 L fermentation of DEM30355, was dissolved in 20 mL of methanol (MeOH), absorbed onto 10 mL of chromatography grade silica and dried. This was loaded onto a 100 g silica SNAP Biotage cartridge and eluted with ethyl acetate  at a flow rate of 50 mL/min and a fraction size of 50 mL. Fractions containing tatiomicin  were combined and the organic solvent was evaporated under reduced pressure. The resulting black oil was dissolved in 5 mL of methanol and loaded onto an LH20 size exclusion column . The column was eluted with methanol at a flow rate of 1 mL/min. The first 80 mL of eluent (1 CV) was discarded, after which 10 mL fractions were collected to give 80 fractions in total. The fractions were analysed by TLC and those containing tatiomicin were combined and the solvent evaporated under reduced pressure. The resulting dark yellow oil was dissolved in 5 mL of methanol, absorbed onto 5 mL of chromatography grade silica, dried and loaded onto a 50 g silica SNAP Biotage cartridge. The column was eluted with diethyl ether at a flow rate of 50 mL/min, with 50 mL fractions collected. The fractions were analysed by TLC and those containing tatiomicin, as a mixture of two epimers, were combined, and the solvent evaporated under reduced pressure to give 171.5 mg of yellow powder.D -78 ; 1H NMR  δH 8.12 , 7.05 , 6.92 , 5.77 , 5.49 , 5.47 , 3.85  3.84 , 2.78 , 1.94 , 1.20 ; 13C NMR  δC 196.3, 174.9, 152.9, 151.2, 133.2, 131.9, 129.0, 124.7, 123.3, 118.9, 115.9, 112.8, 83.5, 82.7, 81.7, 66.5, 56.5, 56.4, 49.8, 15.6, 11.3; HRESIMS m/z found 425.1207, C21H22O8Na requires 425.1207.[α]1H NMR  δH 8.14 , 7.07 , 6.95 , 5.76 , 5.52 , 5.54–5.50 , 3.88  3.87 , 2.81 , 1.98 , 1.41 ; 13C NMR  δC 196.3, 177.7, 153.1, 151.5, 133.9, 132.0, 129.4, 125.8, 123.5, 119.2, 116.1, 113.1, 83.7, 81.9, 80.0, 66.8, 56.8, 56.7, 49.7, 15.9, 11.3.Exponentially growing cells (20 mL) were harvested , lysed and the DNA was purified via a phenol–chloroform extraction. The purity of extracted DNA was assessed via NanoDrop and visualisation on an agarose gel. Sequencing was performed using the next-generation sequencing platforms of Illumina HiSeq 2000 technology (Illumina) and PacBio (Pacific Biosciences). The data received from PacBio sequencing was assembled into 13 contigs using the open source PacBio software SMRTportal and subsequently corrected with the illumina reads using CLC Genomic Workbench 7.0.4 with a trial license. The gene sequence of the tatiomicin gene cluster was analysed using AntiSMASH 6.0.1 The cluster was visualized in artemis. The rishirilide gene cluster was retrieved from the MIBiG database (MGC0001179) and using the Blastp algorithm manually aligned to the tatiomicin gene cluster to find homologous genes Table . The nucAmycolatopsis strain DEM30355 DNA was constructed in pESAC13 (by BioS&T). The supplied genomic library was screened for the tatiomicin gene cluster using PCR with multiple primer pairs . Six strains showed putative tatiomicin production and were subjected to expression analysis at a 500 mL scale in GYMG media in baffled flask for 5 days at 30 °C. One exconjugant showed via TLC clear tatiomicin production. The supernatant was delipified using petroleum ether before extraction of tatiomicin using diethyl ether at equal volume. The tatiomicin extract was then subjected to HPLC and MS analysis. Alternatively, the strain was grown on GYMG agar . The plates were homogenised, freeze thawed and extracted with 500 mL ethyl acetate and subjected to LC–MS analysis.The received exconjugants were arrayed into five 24 well plates filled with 4 mL of GYMG media  and incubated shaking at 300 rpm for 5 days at 30 °C. The supernatant was subsequently freeze dried and resuspended in 100 µL DMSO. This was subjected to TLC analysis in ethyl acetate , 3 isolates acquired from the American Type Culture Collection , 1 isolate from the National Collection of Pathogenic Fungi  and 36 MRSA strains frequently encountered in Europe. Tatiomicin/tatiomicin* (3:1) was prepared at a stock concentration of 10 mg/mL in 100% DMSO, which was tested in IsoSensitest agar at a concentration range of 0.031 to 64 μg/mL, and inoculated using a multipoint inoculator to deliver a final concentration of 104 CFU/1 µL spot of each isolate. An inhibitor-free control plate was also included. Incubation occurred at 37 ± 0.5 °C for 18 h. The MIC was defined as the lowest concentration of tatiomicin/tatiomicin* inhibiting visual growth after overnight incubation.Agar titration was performed in accordance with British Society for Antimicrobial Chemotherapy guidelinesA stock solution of tatiomicin  was prepared in DMSO (10 mL) to give a 2.50 mM solution. A stock solution of L-cysteine hydrochloride  was prepared in deionised water (1 mL) to give 0.01 M solution. A stock solution of L-cysteine methyl ester hydrochloride  was prepared in deionised water (1 mL) to give 0.01 M solution.To a 5 mL pear shaped RBF, was added water (725 µL), 0.01 M solution of L-cysteine hydrochloride or L-cysteine methyl ester hydrochloride in water  and a 2.50 mM solution of tatiomicin in DMSO . The reactions were stirred for 180 min at room temperature. After 30, 60, 90, 120, 150 and 180 min, a 50 µL aliquot of each reaction mixture was diluted into 250 µL of water, and then analysed by LCMS to identify possible Michael addition products.A stock solution of tatiomicin  was prepared in DMSO (1 mL) to give a 25 mM solution. A 4.5 mM solution of LcrV (272–291) was prepared in 200 mM Tris buffer (pH 8) in 20 mM DTT (as a stabilizer).To a 0.2 mL Eppendorf tube, was added water (2.6 µL), 4.5 mM solution of LcrV (271–291) in 20 mM DTT , 200 mM solution of Tris buffer 200 mM (pH 8)  and 25 mM solution of tatiomicin in DMSO . The resulting mixture was mixed for 5 s (vortex) at room temperature. After 0, 15, 30, 45 and 60 min, a 2 µL aliquot of the reaction mixture was taken, diluted with 10 µL water and then analysed by LCMS to examine the presence of Michael addition products.B. subtilis 168CA was grown to an OD600 of 0.1 in LB medium at 37 °C while shaking. Tatiomicin/tatiomicin* was imaged at 2 × the inhibitory concentration at an OD600 0.1 (32 µg/mL). The control antibiotics rifampicin at 3 µg/mL, nisin at 33 μg/mL and gramicidin at 38 µg/mL (mix of gramicidin A–D). We assayed the effect of the compounds after 8 min of incubation with the compounds by transferring the cells onto a microscope slides covered with 1.2% agarose in water. For DiSC3(5) and Sytox Green staining a final concentration of 2 μM and 50 nM was used retrospectively. Cells were incubated for 5 min at 37 degrees before adding the compounds. Microscopy was carried out with Nikon Eclipse Ti (Nikon Plan Apo 1.40 Oil Ph3 objective) and the images acquired with Prime 4.2 sCMOS camera (Photometrics) and Metamorph 7 (Molecular Devices).Supplementary Information.


The composite provides spaces to buffer volume expansion, its hierarchical profile benefits the fast transport of Li+ ions and electrons, and the Ni6MnO8 coating on SnO2 improves conductivity. Compared to SnO2, the Ni6MnO8 coating significantly enhances the discharge capacity and stability. The SnO2@Ni6MnO8 anode displays 1030 mAh g−1 at 0.1 A g−1 and exhibits 800 mAh g−1 under 0.5 A g−1, along with high Coulombic efficiency of 95%. Furthermore, stable rate performance can be achieved, indicating promising applications.Semiconductor-based composites are potential anodes for Li-ion batteries, owing to their high theoretical capacity and low cost. However, low stability induced by large volumetric change in cycling restricts the applications of such composites. Here, a hierarchical SnO The demands of next-generation Li-ion batteries include sufficient energy density and improved safety and cycle life. However, at present, graphite anodes with a low theoretical capacity are dominant, restricting the development of emerging Li-ion batteries ,2,3,4. I2, etc.) ,8,9,10 a2, etc.) ,12,13, m2, etc.) ,19,20,212 has been widely considered one of the most promising anodes among metal dioxides in recent years, owing to its abundance, environmental friendliness, and Li-ion storage performance. Nevertheless, its theoretical capacity (782 mAh g−1) and the volume change of SnO2 in lithiation/delithiation lead to a rapid decrease in capacity. The low conductivity of SnO2 also leads to poor rate performance and low capacity retention. Furthermore, large volume change leads to electrode pulverization [2 hollow sphere, which evenly coated SnO2 with a carbon nanolayer [2@C combining SnO2 with carbon. The hollow sphere ensured structural stability during long-term charge and discharge, exhibiting stability of 1628 mAh g−1 under 0.1 A g−1 [2 combining SnO2 with conductive graphene, which exhibited improved conductivity [SnOrization ,23,24. Lanolayer . The hol.1 A g−1 . Liu anductivity .2O4, ZnFe2O4, CoMn2O4, ZnCo2O4, and ZnMn2O4 exhibit satisfactory electronic conductivity and reversible capacity, which may be caused by the heterovalent cations and corresponding general redox reactions [2O4 microsphere using a carbon template with a solvothermal method [2O4 microspheres buffered the volumetric change in Li+ insertion/extraction, thereby reducing electrochemical pulverization. CoMn2O4-based anode exhibited 1643 mAh g−1 at 0.1 A g−1. Ren et al. presented ZnCo2O4@reduced graphene oxide nanocomposites; ZnCo2O4 nanoparticles were anchored uniformly and compactly on reduced graphene oxide. High specific surface area and mesoporous structure provided a large contact area with an electrolyte and shortened the transferring pathway of the Li+ ions [2O4 nanocomposite, which was conducive to the penetration of the electrolyte during the charge/discharge process [+ in those composites would cause considerable volume change during cycling, resulting in serious agglomeration of the materials and the exfoliation of the electrode materials from the collector, rapidly reducing the performance. Engineering optimal structures to efficiently accommodate the volume change has become an important strategy to improve Li storage properties [In addition, bimetal oxides such as CuCoeactions ,29. Zhanl method . The yolLi+ ions . Chen et process . Howeveroperties ,34.2@Ni6MnO8 composite comprising Ni6MnO8 nanoflakes growing on the surface of a three-dimensional (3D) SnO2, as illustrated in 2@Ni6MnO8 composite was prepared through a two-step hydrothermal method. After calcination, hollow SnO2 was formed, providing space for the penetration of electrolytes, benefitting Li+ ion diffusion and accommodating the volumetric change during cycling. The special structure and composition of composites have rarely been reported. Composite-based anodes achieve positive electrochemical performance. The capacity remains 1030 mAh g−1 after cycling 50 times under 0.1 A g−1 and 800 mAh g−1 under 0.5 A g−1, in addition to high Coulombic efficiency exceeding 95% and recoverable rate performance.Here, we developed a hierarchical SnO4·5H2O  was added to the solution with continuous stirring. After complete dissolution, it was transferred into a PTFE-lined stainless-steel reactor and reacted at 180 °C for 6 h. Then, the obtained precipitate was washed with water and ethanol alternately by centrifugation, then dried in an oven.First, 20 mmol sucrose was dissolved in 40 mL of deionized water. Then, 10 mmol SnCl2/C precursor was ultrasonically dispersed in 40 mL of deionized water. Then, 300 mg of Ni(NO3)2·6H2O, 530 mg of Mn(NO3)2·4H2O, 140 mg of hexamethylenetetramine, and 29 mg of trisodium citrate dihydrate were subsequently added into the above solution under ultrasonic treatment. The solution was placed in the reactor at 140 °C for 6 h. Then, it was taken out and cooled. The sample was washed and dried at 60 °C and annealed at 500 °C for 1 h in air with a heating rate of 5 °C per min.An amount of 0.1 g of the SnOThe samples were characterized on a Bruker D8 X-ray diffractometer (XRD), a Hitachi HT-7700 transmission electron microscope (TEM), an S-8100 scanning electron microscope (SEM), a MICROMERITICS ASAP 2460 Brunauer–Emmett–Teller (BET) instrument, and an ESCALAB 250 X-ray photoelectron spectroscope (XPS).2@Ni6MnO8, carbon black, and carboxymethyl cellulose were mixed (mass ratio = 8:1:1) and coated on Cu foil. After drying at 60 °C in a vacuum overnight, the sample was cut into small discs. 2032-typed cells were assembled by using a glove box . Li foil was used as the counter electrode, and the Celgard polypropylene film was used as a separator. Electrolytes contained 1 M LiPF6 in ethyl carbonate and diethyl carbonate (volume ratio = 1:1). Galvanostatic charge–discharge was tested on a measuring system .A coin cell system was used to measure electrochemical performance. First, 3D SnO2/C precursor, which exhibits a coral-like structure with a diameter of 400–500 nm. The XRD pattern of SnO2/C precursor is presented in 2 (JCPDS card #41–1445), respectively [2@Ni6MnO8 after in situ growth of Ni6MnO8 on SnO2/C precursor. The surface of SnO2/C is completely coated by dense Ni6MnO8 nanoflakes. The 3D profile is well-maintained, indicating a robust structure.ectively ,36. Figu2@Ni6MnO8 calcined at 500 °C. The morphology remains unchanged compared to that before heat treatment. Dense nanoflakes are coated on the surface without breaking. 2 inside. The TEM image shows thin nanoflakes, which are beneficial in terms of providing large active sites for reaction with Li+ ions in Li-ion batteries. The XRD pattern of SnO2@Ni6MnO8 is displayed in 6MnO8 (JCPDS card #49–1295), respectively, which verify a secondary growth. The BET measurements of SnO2@C and SnO2@Ni6MnO8 are shown in 6MnO8 nanoflakes, the BET surface area of SnO2@Ni6MnO8 is as high as 69.6 cm2 g−1 compared to SnO2@C (10.3 m2 g−1). The inserts show that pore distributions of SnO2@C and SnO2@Ni6MnO8 dominate at about 3 nm and 15 nm. The increased surface area and large pore volume promotes electrolyte penetration.2@Ni6MnO8 and the elemental distribution are displayed in An SEM image of SnO2@Ni6MnO8 were measured. The coexistence of Mn, Ni, C, O, and Sn is verified by the XPS survey spectrum  and surface-adsorbed oxygen (531.8 eV) [The XPS spectra of SnOspectrum a. The XPspectrum b is chartrum  ,41,42. F2@Ni6MnO8 nanocomposite and SnO2 were measured from 0.01 to 3.0 V. The cycling performance at a current density of 0.1 A g−1 is presented in 2@Ni6MnO8 anode shows a high specific capacity of 914 mAh g−1. After 50 cycles, the SnO2@Ni6MnO8 anode maintains a reversible capacity of 1030 mAh g−1, and its Coulombic efficiency exceeds 96%. The first-cycle capacity of SnO2 is 876 mAh g−1. However, the capacity decreases rapidly to 509 mAh g−1 after the 50th cycle, indicating that the Ni6MnO8 coating considerably enhances the capacity and stability. The slight increase in capacity is ascribed to the activation of the nanoscale composite. 2@Ni6MnO8 anode. There are two plateaus on the discharge curves, which correspond to Li+ ion insertion in SnO2@Ni6MnO8. In the charging process, there are three plateaus at around 0.5–1.0 V, 1.2–2.0 V, and 2.0–3.0 V, which are assigned to Li+ ion extraction. −1. After cycling 50 times, the SnO2 anode shows a low capacity of 437 mAh g−1. In contrast, the capacity of SnO2@Ni6MnO8 is maintains as high as 800 mAh g−1, with a Coulombic efficiency of about 95%, indicating a sufficient reversibility. 2@Ni6MnO8 anode exhibits a competitive performance, as shown in The electrochemical properties of 3D SnO2@Ni6MnO8 nanocomposite. −1 at 0.1, 0.2, 0.5, and 1.0 A g−1, respectively. Once the rate returns to 0.1 A g−1, the capacity recovers to 760 mAh g−1. The galvanostatic charge–discharge curve for each current density with a similar profile is shown in 2@Ni6MnO8 composite are attributed to the hierarchical morphology with nanoflakes growing in situ on a 3D structure, which provides efficient space to alleviate the volumetric expansion and enable rapid transport of electrons and ions.+ ion insertion in the SnO2/Ni6MnO8 anode. In contrast, the peaks at 0.61, 1.38, and 2.31 V are assigned to the extraction of Li+, in accordance with the charge–discharge curves shown in 2/Ni6MnO8 and SnO2/C anodes. The fitting values before and after cycling are about 130 and 200 Ω, respectively, indicating an improved conductivity after coating with Ni6MnO8. An interesting line of future research would be to obtain the diffusion coefficient through EIS spectra [CV curves are shown in  spectra .2@Ni6MnO8 composite was developed for a secondary battery anode with satisfactory Li-storage performance by the hydrothermal method. The presented approach is controllable and simple; however, we expect that a higher-yield preparation can be achieved in the future. The 3D structure provides a large space to buffer volumetric change during charge and discharge. The hierarchical surface shortens the transferring distance of Li+ ions and electrons, and the Ni6MnO8 coating on SnO2 improves conductivity. Compared to SnO2, the coating of Ni6MnO8 considerably enhances the discharge capacity and stability of the anode. The capacities of SnO2@Ni6MnO8 anodes remain 1030 and 800 mAh g−1 after 50 cycles at 0.1 and 0.5 A g−1, respectively, and the Coulombic efficiency continues to exceed 95%. Moreover, a high and reversible rate performance is also achieved. We believe that the 3D structure presented here and the high electrochemical performance opportunities to engineer emerging composites for secondary battery systems. However, potential challenges should also be considered in future investigations, such as extending the applicable materials, reducing the cost of reagents and steps during synthesis, avoiding the toxicity, etc.In summary, a hierarchical SnO