ATP) channels couple the intracellular ATP concentration to insulin secretion. KATP channel activity is inhibited by ATP binding to the Kir6.2 tetramer and activated by phosphatidylinositol 4,5‐bisphosphate (PIP2). Here, we use molecular dynamics simulation, electrophysiology and fluorescence spectroscopy to show that ATP and PIP2 occupy different binding pockets that share a single amino acid residue, K39. When both ligands are present, simulations suggest that K39 shows a greater preference to co‐ordinate with PIP2 than with ATP. They also predict that a neonatal diabetes mutation at K39 (K39R) increases the number of hydrogen bonds formed between K39 and PIP2, potentially accounting for the reduced ATP inhibition observed in electrophysiological experiments. Our work suggests that PIP2 and ATP interact allosterically to regulate KATP channel activity.ATP‐sensitive potassium  lipids and inactivated by the binding of ATP.The K2 and ATP. A mutation to this residue (K39R) results in neonatal diabetes.K39 has the potential to bind to both PIPThis study uses patch‐clamp fluorometry, electrophysiology and molecular dynamics simulation.2 competes with ATP for K39, and this reduces channel inhibition by ATP.We show that PIP2 by increasing the number of hydrogen bonds with PIP2, when compared with the wild‐type K39. This therefore decreases KATP channel inhibition by ATP.We show that K39R increases channel affinity to PIP Abstract figure legend In this study, we have used electrophysiology, patch‐clamp fluorometry and molecular dynamics simulations to study the dynamic interplay of phosphatidylinositol 4,5‐bisphosphate (PIP2) and ATP in the regulation of the KATP channel, identifying K39 as a residue that engages with both ligands. ATP) channels couple the metabolic state of the pancreatic β‐cell to insulin secretion  subunits, surrounded by four regulatory sulphonylurea receptor 1 (SUR1) subunits , increases the channel open probability   simulation  and increases the predicted PIP2 binding affinity  leads to reduced ATP inhibition  binding to the C‐terminus of Kir6.1 and Kir6.2 channels  simulations to determine the dynamics of K39 when both ATP and PIPATP propeller channel octameric complex consisting of four copies of Kir6.2 (residues 32−352) and four copies of SUR1 (PDB entry: 6BAA). These complexes were converted to a coarse‐grained representation using martinize.py, embedded in a palmitoyl‐oleoyl‐phosphatidylcholine (POPC) bilayer and solvated in water and 0.15 M NaCl using a self‐assembly MemProtMD pipeline . All simulations were carried out using the Martini v.2.2 biomolecular forcefield  the human Kir6.2 model from residue 32 to 352 without SUR1 in the propeller conformation (Protein Data Bank (PDB) entry: 6BAA)  was converted to atomistic using the CG2AT pipeline , and simulations were carried out for three repeats.The coarse‐grained simulation system  calculations and distance calculation were carried out using GROMACS‐2019.4  at 37°C, 5:95 CO2:air. Cells were seeded in T25 flasks for 24 h, before transfection with TransIT‐LT1 . Protein expression and trafficking to the plasma membrane were optimized by including 300 μM tolbutamide in the transfection media  or poly‐l‐lysine‐coated 35 mm Petri dishes (Corning). pCDNA4/TO and pANAP were obtained from Addgene. peRF1‐E55D (Homo sapiens) and pCGFP_EU (Aequorea victoria) were kind gifts from the Chin Laboratory  and Gouaux Laboratory , respectively.Human Kir6.2 and SUR1 were subcloned into pCGFP_EU [green fluorescent proten (GFP)‐tagged constructs] and pcDNA4/TO, respectively. Site‐directed mutagenesis and amber stop codons were introduced using the QuikChange XL system  and verified by sequencing , as previously described  solutions contained (mM): 140 KCl, 1 EGTA and 10 Hepes (pH adjusted to 7.3 with KOH). The intracellular (bath) solutions contained (mM): 140 KCl, 1 EDTA, 1 EGTA, 10 Hepes (pH adjusted to 7.3 with KOH). Inside‐out patches were excised from transfected HEK293T cells using borosilicate glass pipettes  pulled to a resistance of 1–3 MΩ. Data were acquired at a holding potential of −60 mV using an Axopatch 200B amplifier and a Digidata 1322A digitizer run through pClamp 9 software . Currents were low‐pass filtered at 1–5 kHz and digitized at 10–20 kHz.2 at the end of each recording at a holding potential of +60 mV. Current inhibition data were fitted with the following Hill equation:Patches were perfused with an eight‐channel μFlow or a manual gravity perfusion system. Different concentrations of ATP (Sigma) or fluorescent trinitrophenyl adenosine triphosphate  were added to the bath solution to assess current inhibition and/or nucleotide binding. Nucleotide‐induced inhibition was corrected for rundown by alternating test concentrations of nucleotide solution with nucleotide‐free solution. The inhibition was expressed as a fraction of the control currents before and after the test solution, as described previously  package in R as a mixed‐effects model  as a result of making the indicated mutation.Each model was run across four chains for 4000 iterations including a burn‐in period of 2000 iterations for a total of 8000 samples. Each model parameter achieved a minimum effective sample size of 5000 and a potential scale reduction statistic (R̂) of 1.00. Contrasts were calculated by subtracting the full posterior probability for the IC−1 grating. Images were collected with 1 s exposures on a Pixis 400BR_eXcelon CCD (Princeton Instruments). Spectra were corrected for background fluorescence, then ANAP intensity was calculated by averaging the fluorescence intensity measured between 469.5 and 474.5 nm. This intensity was corrected for bleaching with a single exponential decay.Fluorescence spectra from excised patches were collected and analysed as described previously  and the 95% intervals (shaded area) of the posterior probability distribution. The Monod–Wyman–Changeux (MWC)‐type model fits are also displayed as the median fit (continuous line) and the 95% intervals (shaded area) of the posterior probability distribution. Where posterior probability distributions for parameter values are shown, they are displayed with the 50, 80 and 95% intervals of the distribution in progressively lighter shades of colour.The MWC‐type model fitted here is described by the following sets of equations:L represents an equilibrium constant where the KATP open probability (Popen) is equal to L/(L + 1), reflecting the ability of KATP to open and close in the absence of nucleotides. Each ligand binding event (KA) is independent, and each bound ligand favours the closed state by the same factor (D). This model is fitted to the combined current inhibition and fluorescence quenching data using the brms package in R  of 1.00.2 binding sites of Kir6.2.In this study, we used both atomistic molecular dynamic simulations and functional studies to explore the relationship between the ATP and PIP2 and ATP binding sites in the Kir6.2 tetramer in the absence of SUR1 in order to exclude ATP interactions with SUR1. Previous simulations have shown that there is no difference in the PIP2 binding site when SUR1 is present  showed slightly less Cα rearrangement than the Apo state. This suggests that the three‐dimensional structure of the protein is highly stable throughout our simulation and might be further stabilized by the ligand . Comparison of the cryo‐EM structure of the ATP binding site with the predicted PIP2 binding site suggests that they lie ∼25 Å from one another  in ATP contacts occurred at residue K39 only when PIP2 was present  is associated with transient neonatal diabetes  had an estimated IC50 value for ATP inhibition of 23.5–43.7 μM . We observed that substitution of K39 by either arginine (Kir6.2‐K39R+SUR1) or glutate (Kir6.2‐K39E+SUR1) resulted in an increase in the estimated IC50 value for ATP inhibition, whereas substitution by alanine (Kir6.2‐K39A+SUR1) had no effect  into Kir6.2 and measured ATP inhibition of Klls Fig. A. To faATP currents  also leads to a reduction in sensitivity to nucleotide inhibition, and substitution to K39A (no charge) does not affect inhibition at all. These findings are at odds with our hypothesis on the importance of hydrogen bonding at this residue. Given that our simulation data suggest that K39 also co‐ordinates ATP binding, we considered the possibility that we might be observing a mixture of effects on both ATP and PIP2 co‐ordination, explaining why we do not observe a lower IC50 for ATP inhibition of this construct. Unfortunately, although the IC50 for ATP inhibition of Kir6.2*‐K39A+SUR1 was indistinguishable from the IC50 for ATP inhibition of Kir6.2*+SUR1 , in support of previous work , supported by EPSRC (grant no. EP/R029407/1).T.P. and S.U. hold a Wellcome Trust OXION studentship. T.P. holds a Clarendon scholarship. Research in P.J.S.’s laboratory is funded by Wellcome (208361/Z/17/Z), the MRC (MR/S009213/1) and BBSRC . Research in F.M.A.’s laboratory is funded by the MRC (MR/T002107/1) and BBSRC . This project made use of time on ARCHER and JADE granted via the UK High‐End Computing Consortium for Biomolecular Simulation, HECBioSim (Statistical Summary DocumentClick here for additional data file.Peer Review HistoryClick here for additional data file.


To further expand our understandingof the chemical and magnetic properties of the Sr2Crx3–MnxAs2O2 solid solution, Sr2Cr2MnAs2O2 has been synthesized. The compoundcrystallizes in the I4/mmm tetragonalspace group with a refined stoichiometry of Sr2Cr1.85Mn1.15As2O2. The M(2) site withinthe M2Pn2 slab is occupied by 42.7% Cr and 57.3%Mn, and the magnetic moments order antiferromagnetically below TN(M2) = 540 K with a C-type antiferromagnetic structure. TheM(1) site within the MO2 layers is fully occupied by Cr,and antiferromagnetic order is observed below TN(M1) =200 K. Along c, there are two possible interplanararrangements: ferromagnetic with the  propagation vectorand antiferromagnetic with the  propagation vector.Magnetic phase separation arises so that both propagation vectorsare observed below 200 K. Such magnetic phase separation has not beenpreviously observed in Sr2M3As2O2 phases  and shows that there are several competingmagnetic structures present in these compounds.Layered Sr 2M3As2O2-type oxypnictides are composed of tetrahedralM2Pn2 and square planar MO2 layers,the building blocksof iron-based and cuprate superconductors. Sr2Cr1.85Mn1.15As2O2 is antiferromagneticand semiconducting. Surprisingly magnetic phase segregation is observedin the oxypnictide Sr2Cr1.85Mn1.15As2O2 because of competing magnetic exchangeinteractions along c.Layered Sr Their superconductivity originates from the iron arsenidelayers,2M2M’As2O2 are especially interesting, as they contain key structural elementsof both the iron arsenide and cuprate superconductors. These materialscrystallize in the tetragonal crystal system, space group I4/mmm, and are composed of square planarM(1)O2 layers and tetrahedral M(2)2As2 layers. These layers are stacked on top of each other perpendicularto the crystallographic c axis and are separatedby layers of alkali-earth cations (A2+). Several oxyarsenides,specifically A2Mx3–M’xAs2O2 ,have been synthesized and investigated. While Sr2Fe2CuAs2O2 should be the most interestingcompound to study, being composed of the cuprate (CuO2)and iron-based (Fe2As2) superconductor superconductinglayers, it was reported that syntheses did not yield a single-phaseproduct.19 However, it was discovered thatCrAs becomes superconducting under pressure as the antiferromagneticordering is quenched22 and that superconductivitycould potentially be induced in Sr2Cr3As2O2, either with pressure or with chemical doping.17 It was also determined that MnP becomes superconductingat ∼8 GPa.23 This prompted our explorationof compounds within the series Sr2Crx3–MnxAs2O2.The oxyarsenides A2Mn3As2O2,19 Sr2Cr3As2O2,16 and Sr2Cr0.77Mn2.23As2O2.18 Sr2Cr3As2O2 exhibits antiferromagneticordering of the M(2) sites below 590.3 K, with the propagation vectork = , and the moments aligned parallel to the c axis. Below 291 K, the M(1) sites order antiferromagnetically, witha K2NiF4-type antiferromagnetic structure andpropagation vector k =  , which causes a spin-flop of the M(2) spins onto the ab plane.16 The Sr2Mn3As2O2 compound exhibits G-typeantiferromagnetic ordering with the propagation vector k = 0 of theM(2) sites below 340 K with spins aligned parallel to the c axis and short-range two-dimensional magnetic correlationsbelow 75 K so that the M(1) sublattice does not exhibit long-rangemagnetic order down to 4 K. Additional peaks were observed at 4 Kbut could not be satisfactorily modeled.19 The mixed transition-metal compound Sr2Cr0.77Mn2.23As2O2 exhibits G-type antiferromagneticordering of the M(2) sites with k = 0 between 410 and 167 K , and antiferromagneticordering of the M(1) sites below 167 K  with k = , which causes a reorientationof the M(2) moments to a C-type antiferromagnetic arrangement  with k = .18Very different magnetic structures have been reportedfor SrN decreasingas the Cr:Mn ratio decreases.19 Xu et al.15 have recently discussed the interlayer couplingof magnetic sublattices in Sr2Cr3As2O2, prompted by their observation of a spin-flop transitionof the M(2) site magnetic moments upon the emergence of long-rangeorder of the M(1) site moments, which was also reported by Liu etal.16 A similar (field-induced) phenomenonhas also recently been observed in EuMnBi224 and coupling of the magnetic sublattices has been observedin the 1111-type compounds such as SmFeAsO,25 CeMnAsO,26 and PrMnSbO.27 However, in these examples, both magnetic sublattices havethe same propagation vector. The coupling of magnetic lattices withdifferent propagation vectors should not occur in principle; however,it seems that A2M2M’As2O2 oxypnictides exhibit this phenomenon.15 The spin reorientation and magnetic coupling were attributedto an antisymmetric exchange ,however, no significant accompanying structural distortion could bedetermined.15The magnetic transition temperatures inthese compounds are alsoaffected by the transition-metal stoichiometry with T2Cr0.77Mn2.23As2O2,18 we have started to explore the magneticstructures of other members of this solid solution series to betterunderstand the interactions between the layers. We have now synthesizedSr2Cr2MnAs2O2, and herewe report the crystal structure and changes in the magnetic structurewith temperature.Since we have observeda similar spin-flip transition in Sr2Cr2MnAs2O2 was prepared using astandard solid-state synthesis on a 1.2 g scale. The starting materialsused were Mn , Cr , As , and SrCO3 . SrO was preparedby heating SrCO3 at 1250 °C for 12 h, followed byquenching and immediate use. Stoichiometric amounts of Mn, Cr, andAs were mixed with a nonstoichiometric amount of SrO (2% deficiency).In agreement with the reported syntheses of Sr2Cr3As2O217 and Sr2Cr0.77Mn2.23As2O2,18 we have observed that the deficiency increasedphase purity. All starting materials were mixed and ground in an inertatmosphere  environment using an agatemortar and pestle. The mixture was pelleted and inserted into a Tacrucible , which was sealedin a quartz tube under vacuum. The pellets were heated at 1080 °Cfor 72 h twice, with intermediate regrinding.A polycrystalline sample of thenominal stoichiometry SrRoom-temperaturepowder X-ray diffraction was performed on a PANalyticalEmpyrean Powder diffractometer. Patterns were recorded using Cu Kα1radiation, in the range of 5° < 2θ < 100°, witha step size of 0.01313°.Variable temperature neutron diffractionexperiments were performedon the D1B and D2B diffractometers at the Institut Laue-Langevin inGrenoble, France (ILL). Approximately, 1.5 g of the sample powderwas inserted into a 9 mm vanadium can. Data were recorded on the D1Bdiffractometer with λ = 2.512 Å, with a temperature rangeof 1.5–420 K upon heating at the rate of 1 K/170 s and 375–673K on heating at the rate of 1 K/200 s, separated into 600 s snapshots. Further patterns were recordedon the D2B diffractometer with λ = 1.594 Å at 1.5, 50,100, 150, 200, 250, 300, 350, 400, 450, 500, and 600 K. Data werecollected for 2.5 h at each temperature step.The temperaturedependence of the magnetic susceptibility was performedwith a Quantum Design Superconducting Quantum Interference Device(SQUID) magnetometer. Field-cooled (FC) and zero-field-cooled (ZFC)measurements were recorded in a field of 1000 Oe between 4 and 340K. The temperature dependence of the electrical resistance was performedwith a Quantum Design physical property measurement system (PPMS).Measurements were recorded on heating between 4 and 340 K.2Cr2MnAs2O2 crystallizesin the tetragonal crystal system with the space group I4/mmm. The unit cell of Sr2Cr2MnAs2O2 is composed of M(1)O2 planesand M(2)2As2 layers, separated by Sr2+ ions, as shown in 2Cr3As2O2 crystal cell,17 modified to Sr2Cr2MnAs2O2 by replacing half ofthe M(2) site Cr occupancy with Mn. The patterns could be indexedwell on the tetragonal unit cell, with cell parameters a = 4.0428(1) Å and c = 18.9578(6) Å. Asmall impurity phase of Sr2Cr2AsO3 is observed and fitted (0.7% by mass). This impurity has previouslybeen identified by Xu et al. during the synthesis of Sr2Cr3As2O2.15Sr15 and the M(2) sites are in tetrahedral coordination.Powder neutron diffraction data were recorded to determine site occupanciesand magnetic ordering of Sr2Cr2MnAs2O2. Rietveld refinement was first performed on the I4/mmm model with the GSAS/EXPGUI29 software using the high-resolution diffraction pattern obtainedat T = 600 K (I4/mmm space group with a = 4.06066(4) Å and c = 19.1220(4) Å (χ2 = 2.91). Theatomic displacement parameters were modeled isotopically with theUisos for Cr and Mn constrained to the same value. Siteoccupancies were refined for all atoms and fixed to 1.0 where therefined values were within ±1% of the full occupancy. It wasdetermined that the M(1) site is fully occupied by Cr, while the M(2)site is occupied by 42.7(4)% Cr and 57.3(4)% Mn. These values wereused, with no further refinement, at all temperatures. This distributionwas attributed to the effects of crystal field stabilization energyand Jahn–Teller distortion, as previously reported for Sr2Mn2.23Cr0.77As2O2.18 The Cr2+ ion favors octahedraland square planar coordination30 and fillscompletely the square planar M(1) site. In contrast, the Mn2+ ion displays no site selectivity and is therefore found at the M(2)site. This is also observed in A2MnZn2As2O2 , where the Mn2+ ionsoccupy the M(1) site and the Zn2+ ions occupy the M(2)site,31 due to the tetrahedral site preference30 of Zn2+. Additional Rietveld refinementsagainst selected patterns are shown in S1, and therefined atomic parameters, cell parameters, and agreement factorsfrom Rietveld fits are shown in Table S1.The M(1) and M(2) transition-metal sites, locatedat Wyckoff positions2a and 4d, respectively, have different local environments. The M(1)sites are in square planar or, arguably, distended octahedral coordination, = 600 K 2a, where2Cr1.85Mn1.15As2O2 sothat, like Sr2Cr0.77Mn2.23As2O2,18 the final synthesizedcompound contains less Cr than expected. The unit cell parametersexhibit normal thermal expansion . Due to the anisotropic shape of the unit cell, thermal expansionfavors the c axis. The unit cell therefore elongatesslightly with increasing temperature, and the c/a ratio changes from 4.68316(2) at 1.5 K to 4.70907(2) at600 K (Table S1). The M(2)–As andCr–O bond lengths are shown in Figure S3. Both bond lengths increase upon heating, while the As–M(2)–Asangles do not change significantly (Table S2). There is also no evidence of magnetostriction at any temperature.Rietveld refinement shows that the stoichiometryis SrN(M2) = 540 K in both the D2B data , which corresponds toa C-type antiferromagnetic arrangement of the M(2) spins with spinsaligned parallel to the c axis. This magnetic structureis the same as that previously reported for Sr2Cr0.77Mn2.23As2O218 at a low temperature (S4). Such peaks are reminiscent of short-rangemagnetic correlations, as observed in Sr2Mn3As2O2 below 75 K,14 in Ba2Zn2MnAs2O2 below30 K,31 and in the pnictide layer of LaMnAsOfrom 360 (1) to 650 (10) K.11 Upon coolingbelow 200 K (TN(M1)), sharper magnetic peaks are observed,which could be indexed by two propagation vectors. The magnetic peakscan be indexed by k2 = , which correspondsto a K2NiF4-type antiferromagnetic structureof the M(1) site magnetic moments with moments ordered parallel tothe c axis, as shown in 3 =   3 and 4. 2NiF4-type magnetic structure. The intraplanar (ab plane) ordering of the magnetic moments is antiferromagnetic.Dueto the body-centered tetragonal crystal structure of the compound,the coupling of the nearest neighboring MO2 layers’magnetic moments is a net zero, as each atom has an equal number offerro- and antiferromagnetically aligned neighbors. The interlayercoupling thus occurs between the next nearest neighboring (nnn) layers,which is favorable in these materials.31 Along c, there are two possible nnn interplanararrangements: ferromagnetic with a propagation vector of  (The moments on the M(1) site order with a K 1/2,0) 5b andan 1/2,0) 5c.2 =   % k2 =  and 71(4)% k3 =  at 168 K, with no further changes down to thelowest temperature measured. During refinement, the fractions of bothphases were constrained with the assumption that the entire volumeof the sample exhibits ordering of the M(1) site moments. Due to theunstable nature of refining both the magnitudes and the fractionsof the separate phases concurrently, they were refined separately,iterating between refining moments and scales in several steps untilsufficient convergence was achieved.Below 200K, a successful fit to the experimental data was achievedwith a model containing 46(4)% k 1/2, 0) 5b and 54/2, 1/2) 5c, with 2Cr1.85Mn1.15As2O2 with temperature is shown in 32 and Ca3(Rux1–Tix)2O7 wheretwo competing antiferromagnetic phases are observed in a narrow dopingrange (x = 0.02–0.05).33 Such magnetic phase separation has not been previouslyobserved in Sr2M3As2O2 phases . It has been established that the intraplanarcoupling constant is several orders of magnitude greater than theinterlayer coupling constant, due to the long interlayer distancein these compounds. As such, interlayer coupling occurs once the correlationlength within the layers reaches a sufficient length.34 The exchange energies for nnn layer ferromagnetic and antiferromagneticalignment along c at the M(1) site would appear tobe almost equivalent in this compound so that there are competingspin structures and two propagation vectors observed below TN(M1). Similar magnetic phase separation has previously been reportedfor RuSr2Y1.5Ce0.5Cu2O10−δ35 and Rb2MnF4.36The variation of the M(1)and M(2) site magnetic moments of SrB, respectively. It haspreviously been shown that there is a correlation between TN at both the M(1) and M(2) sites with the Mn(1)–O and theMn(2)–Mn(2) distances in A2Mn3Pn2O2 ;32 both magnetic ordering temperatures increase as the a cell parameter decreases as a result of increasing superexchange(M(1)–O–M(1) and M(2)–As–M(2)) and directexchange (M(2)–M(2)) interactions in the M(1)–O2 and M(2)2As2 layers. Figure S5 shows that as x is increased inthe Sr2Crx3-MnxAs2O2 solid solution,there is a linear increase in the cell volume. As a result, thereis a clear reduction in the antiferromagnetic ordering temperatureat both the M(1) and M(2) sites as x increases O2 layer composition (CrO2). While the TN inthese compounds differs, the observed short-range magnetic order inSr2Cr1.85Mn1.15As2O2 emerges at a similar temperature (∼290 K), as thelong-range magnetic order in Sr2Cr3As2O2 (291 K). As the cell volume then decreases to 309.194(7)Å3 at 200 K, the correlation length in the CrO2 layers in Sr2Cr1.85Mn1.15As2O2 reaches a sufficient length for the long-rangeorder to be established.At 1.5 K, the saturated magnetic moments of theM(2) and M(1) sitesare 2.86(2) and 3.57(5) μ2+ and Mn2+  in all members of theseries. However, the magnitudes of the M(2) and M(1) site momentsboth rise as the Mn2+ occupancy increases. The magnitudeof the M(1)- and M(2)-ordered moments increases almost linearly with x in Sr2Crx3–MnxAs2O2, for 0≤ x ≤ 2.23 so that there does not seemto be any effect of Mn2+/Cr2+ disorder on theoverall ordered moment. The results suggest that the M(2) site momentmagnitude is directly dependent on the composition of the layer, asthe magnitude increases in proportion to the concentration of Mn2+ in the M(2)2As2 layer. The M(1) momentalso increases linearly with x, which is surprisinggiven that there is no change in the M(1)O2 layer compositionfrom x = 0–1.15. This increase in the M(1)moment could be a result of the reduction in covalency as the M(1)–Obond length increases across the series.Due to the partially covalent nature of the M–Asand M–Obonds, the magnitudes of the saturated magnetic moments are lowerthan the theoretical values for Cr2Crx3–MnxAs2O2 seriesare all different.18 Sr2Cr3As2O2 and Sr2Cr1.85Mn1.15As2O2 both exhibit C-type antiferromagneticorder in the M(2)2As2 layer below TN(M2), whereas G-type antiferromagnetic order is observed for Sr2Mn2.23Cr0.77As2O2 and Sr2Mn3As2O2. Thiswould suggest that the presence of Cr in the M(2)2As2 layer results in ferromagnetic interactions along c. As the order is established on the M(1) site below TN(M1), different magnetic structures are observed. In Sr2Cr3As2O2, the ordering ofthe M(1) site moments causes a spin-flop of the M(2) site moments.15 In Sr2Cr0.77Mn2.23As2O2, the ordering of the M(1) moments isfollowed by a spin-flip of the M(2) moments, shifting the antiferromagneticordering from G-type to C-type. In Sr2Cr1.85Mn1.15As2O2, there is no evidenceof a spin-flip or -flop so there does not appear to be any couplingof the M(1) and M(2) spins. This could be driven by the sizeable cationdisorder in the M(2)2As2 layer, but furtherresearch is warranted. In contrast to other members of the series,magnetic phase segregation is observed in Sr2Cr1.85Mn1.15As2O2 below 200 K and thetwo phases remain in competition down to the lowest measured temperature.The magnetic structuresreported so far for the Sr2Cr1.85Mn1.15As2O2 is presented in 2As2 layer at TN(M2) = 540 K. There is no evidenceof any further magnetic transitions in the susceptibility. The low-temperaturemagnetic transition in Sr2Cr0.77Mn2.23As2O2 was also not evident in the magneticsusceptibility data.18 There is no divergencebetween the FC and ZFC susceptibilities, and the upturn in the susceptibilitybelow T ≈ 100 K is attributed to paramagneticimpurity spins.The temperature variation of the magnetic susceptibility of Sr2Cr1.85Mn1.15As2O2 ispresented in –4 Ω cm, which is suggestive of ahighly doped semiconductor verging on metallic behavior. The resistivitycan be fit to the Arrhenius equation between 120 and 225 K givinga band gap of 2.85 meV. The electronic properties of the A2M2M’As2O2 compounds are dictatedby the M(2)2As2 layers.18 Cr2As2 layers exhibit metallic conductivity,17 while Mn2As2 layers exhibitsemiconducting behavior.18 In the caseof Sr2Cr1.85Mn1.15As2O2, the M(2)2As2 layers are composed ofMn1.15Cr0.85As2 and its resistivityexhibits a highly doped semiconductor-like temperature dependence.The conductivity of the Sr2Crx3–MnxAs2O2 phasesincreases as the percentage of Cr on the M(2) site rises18 so that the electronic properties can be tuned by varying the ratioof Cr:Mn on the M(2) site.The thermal variation of the electrical resistivityof Sr2Cr1.85Mn1.15As2O2, which is the fourth member of the Sr2Crx3–MnxAs2O2 solid solution. The magnetic properties are distinct fromthe other members of the series. In Sr2Cr1.85Mn1.15As2O2, below TN(M1), magnetic phase separation is observed so that the M(1) momentsorder with the propagation vectors k2 =  andk3 = . The two phases remain in competitiondown to the lowest measured temperature. The competing spin structuresarise as the exchange energies for nnn layer ferromagnetic and antiferromagneticalignment at the M(1) site along c are almost equivalentin this compound. The results show that it is possible to tune themagnetic transition temperatures and magnetic structures by dopingon the Cr site in Sr2Crx3–MnxAs2O2.Substitution of Sr2+ for Ca2+ and Ba2+ or a variable temperature neutron diffraction at high pressure couldprovide further insight into the effects of interlayer distances andcell size on magnetic ordering in the Sr2Crx3–MnxAs2O2 compounds.We have synthesized and analyzed the crystaland magnetic structuresof Sr


FTIR spectroscopy of the unfolding structure of egg white protein revealed changes in the protein’s secondary structure, such as the development of β-sheets and β-turns, as well as the binding of negatively charged phosphate groups on the serine, threonine, and tyrosine side chains. The phosphorylation and microwave treatments reduced the particle size of the egg white protein powder while increasing the surface area of the protein molecules, according to SEM analyses.The effects of phosphorylation pre-treatments at 1.5, 2.5, and 3.5% levels, as well as microwave application at 200, 400, and 700 watts levels for 2 min, on the functional parameters of egg white powder obtained by the freeze dryer procedure were investigated. P1.5-M200 had the highest oil-holding capacity, emulsion stability, and emulsion activity, while P2.5-M200 had the highest foam capacity. The P2.5-M400 had the largest particle size, and P3.5-M200 had the highest degree of phosphorylation and protein solubility. On the other hand, P3.5-M200 had the highest solution viscosity by 1% ( Egg white contains a variety of proteins, including ovalbumin, ovotransferrin, ovomucoid, ovomucin, and lysozyme . Eggs arProtein phosphorylation is a typical post-translational protein modification occurring in nature. In general, amino acid residues are phosphorylated by the addition of a covalent phosphate group bound by the protein kinase by serine, threonine, and tyrosine. Phosphorylation modifies the amino acid side chain by adding a charge and a hydrophilic group. According to various studies, phosphorylated proteins have higher solubility than non-phosphorylated proteins because they have a larger negative charge ,8. The p3PO4)) and investigated their physicochemistry and rehydration behavior.For a few years, microwave-assisted chemical procedures have been employed to achieve chemical reactions. When compared to traditional heating techniques, chemical synthesis with microwaves help can significantly shorten reaction duration while also improving yield and product purity. Microwave-assisted approaches have previously been used by certain researchers in the chemical alteration of dietary protein . When coThere are several drying techniques available for producing sodium tri-phosphate egg white powder (STP-EWP), and these drying methods should be chosen based on drying efficiency and dried product quality. It has been found that freeze-dried proteins retain their natural structural shape better because they are subjected to fewer heat and water evaporation-related stresses. The freeze-dried particles are more porous and shrink less. According to Lili et al.  freeze-d2HPO4 , microwave treatment with power , and finally freeze drying (0.04–0.06 mbar & −50 °C) on the functional characteristics including protein solubility, foam capacity, foam stability, emulsion activity, emulsion stability, water-holding capacity, oil holding capacity, viscosity of solution 1%, as well as degree of phosphorylation, particle-size distribution, FTIR phosphorylation analysis, and SEM morphological analysis of egg white protein powder were investigated.For the first time, the combined effects of phosphorylation with Na2HPO4), citric acid dihydrate, nitric acid, sulfuric acid, perchloric acid, trichloroacetic acid, potassium bromide, bovine serum albumin, and the rest of the reagents .A total of 360 fresh chicken eggs were provided from a local farm . The analytical-grade ingredients were disodium hydrogen phosphate  treatments .−1 mixed solutions  at pH 8 by lowering the pH with citric acid dihydrate . Then the solutions were microwaved for 2 min with powers of 200, 400 and 700 watts. The solutions were immediately suspended in cold water at 0 °C for 4–5 min in order to prevent excessive reactivity and bring the temperature of the solution up to 10–15 °C. The extra phosphates were then precipitated as calcium phosphate by adding 19, 32, and 44 g L−1 of calcium chloride to 1.5, 2.5, and 3.5% phosphorylated solutions, respectively. To remove excess calcium phosphate, the solutions were centrifuged at 3000× g for 5 min at room temperature. At a temperature of −50 °C and an air pressure of 0.04–0.06 mbar, the supernatant was dried in a freeze drier [DSP was dissolved in the egg white protein solution to generate 15, 25, and 35 g LA Brookfield rotating viscometer with a cylindrical spindle  (LV-61) was used to evaluate the viscosity of PM-EWP samples (1%). The spindle revolved at 60 rpm. An aliquot of 50 mL was added to the 80 mL flask before testing and allowed to equilibrate at 25 °C .g for 30 min at 4 °C. Protein solubility was calculated using grams of soluble protein per 100 g of egg white protein powder.With minimal adjustments, the technique of Salvador et al.  was used−1 solution) was put in a graduated glass cylinder  and whipped for 4 min with a laboratory homogenizer at 9500 rpm . The propeller was removed immediately after whipping and the glass cylinder was sealed with parafilm to minimize foam disruption. The foam capacity (FC) was computed by taking the foam volume after whipping (mL) at 0 min and calculating it to a percent of the original foam volume (mL). Stefanovi et al. [A 50-mL aliquot of PM-EWP solution  was made with sunflower oil in a 1:1 molar ratio and centrifuged for 5 min at 1100× g. Emulsion activity was calculated as the ratio of the height of the emulsified layer to the total content in the tube using Equation (3). Equation (4) was used to calculate the emulsion’s stability after heating it at 80 °C for 30 min and centrifuging it at 1100× g for 5 min [PM-EWP dispersion (1% or 5 min .(3)Emulg for 30 min. The g of water retained per gram of material was used to compute the water-holding capacity. In grams of oil retained per gram of protein sample, the oil-holding capacity was reported [First, 1 g of PM-EWP was weighed and stirred for one minute in 10 mL of distilled water or maize oil to determine these properties . The supernatant volume was calculated after centrifuging the protein suspensions at 2200× reported .-1 sample solution was mixed with the same volume of 15% trichloroacetic acid and centrifuged at 3000× g for 20 min. The quantity of phosphorus bound to proteins was calculated using the difference between total phosphorus and Pi content [The phosphorus content of the digest was utilized to compute the total phosphorus content of the protein, which was digested in nitric acid, sulfuric acid, and perchloric acid (10:2:1). To measure inorganic phosphorus (Pi), 5 mL of 10 Mg/mL content .s was measured using laser light scattering on a Malvern Mastersizer 2000 equipped with a Scirocco 2000 dry dispersion unit . Moderate particles were added to dry the dispersion device to achieve the required opacity, and the PSD was determined at least three times. Percentile values are represented by the letters d(10), d(50), and d(90). These are statistical characteristics that may be derived directly from the particle size distribution’s cumulative distribution. They represent the particle size below which 10%, 50%, or 90% of all particles are detected. D(4.3) is the volume-weighted mean size. It is the mean diameter, which may be calculated directly from particle size measurements when the recorded signal is proportional to the particle volume. Surface-weighted mean size, i.e., d(3.2) is defined as the diameter of a sphere that has the same volume/surface area ratio as a particle of interest [The PSD of the PM-EWPinterest ,19.−1 (Nicolet Nexus 470). The instrument’s resolution was 4 cm−1, and each sample was scanned using air as the backdrop. In total, 32 scans were used to create each measurement. The materials were mixed with potassium bromide and formed into pellets before being measured [At 25 °C, each sample was evaluated using an FTIR spectrometer in the wave number range of 500–4000 cmThe morphologies of the EWP were imaged using a Tungsten-filament SEM . Prior to SEM, the samples were coated with around 20 nm gold–palladium using a gold sputter module in an argon-filled high-vacuum evaporator. The accelerating voltage was set to 15 kV, and the sputtering period was set to roughly 30 s .p < 0.05) using statistical SPSS software version 16 .This study utilized a completely randomized design with two variables. The first variable was the disodium hydrogen phosphate proportion at 1.5, 2.5, and 3.5%, while the second was the microwave device power at 200, 400, and 700 W for 2 min. Three control samples were used: one with no phosphorus and no microwave treatment, one without phosphorus and 700 W microwaves, and one with 3.5% phosphorylation but no microwave treatment. As a consequence, 12 different treatments were investigated. Each test was replicated three times. Physicochemical studies were performed on the first day of PM-EWP manufacturing. Duncan’s multiple range test revealed significant differences at the 95% confidence level (p < 0.05) as compared to the control (untreated) sample, i.e., P0-M0. Protein solubility is the most significant functional characteristic of a protein due to its influence on emulsifying, foaming, and gelling qualities [ualities . This phualities ,23. Too-ualities .Increasing the microwave power from 400 to 700 watts greatly enhanced the stability of the foam at each level of phosphorylation %, although the difference between different levels of microwave power of 200 and 400 watts was not significant. At all the same levels of applied microwave power, 3.5% and 2.5% phosphorylation showed the highest and lowest foam stability, respectively. P0-M700 and P3.5-M0 treatments had the highest and lowest foam stability, respectively. According to the statistics, the group with the greatest foaming capacity did not have the best foaming stability. Excessive microwave power might be to responsible for the findings. According to Duan et al. , extensip < 0.05), the water-holding capacity increased. The water-holding capacity of the control sample, P0-M0, was greater than that of the microwave-assisted phosphorylation samples (p < 0.05). p < 0.05). The oil holding capacity of the control sample, P0-M0, was greater than that of the microwave-assisted phosphorylation samples (p < 0.05). The water-holding capacity and wetting behavior were affected by particle size and surface tension, with the smaller particles being less likely to be wetted. The PM-EWP samples exhibited a more porous structure, but the lowest particle size and highest specific surface area [The water-holding capacity of EWP under various conditions is shown in ace area .Phosphorylation modification might prevent further exposure of the hydrophobic groups by rearranging them inside the protein molecules , which cw/v) under various conditions is shown in p > 0.05). At varying shear rates, the viscosity of egg white power solution in the control group was essentially the same, demonstrating Newtonian fluid properties. All egg white powder solutions following microwave treatment exhibited shear thinning behavior, indicating non-Newtonian fluid properties. This disparity might be explained by the following two factors: first, in the control group, protein molecules with a tight and regular structure were largely dispersed in the same flow layer of solution, and there was no evident entanglement between them. Second, the flexible protein molecules were placed in distinct flow levels of solution and entangled after microwave treatment. This effect was also previously observed by other authors [The viscosity of PM-EWP solution 1% ( authors . The apperaction .p < 0.05). Dry heating at 2 °C and pH 1 for 2 days in the presence of pyrophosphate enhanced the amount of phosphorus in egg white protein, soluble protein in soybean, ovalbumin, and ovotransferrin, according to Hayashi et al. [68 and Ser344. The list of amino acids that can be phosphorylated has been expanded to include Ser, Thr, Tyr, Arg, and Lys. Since egg white contains naturally phosphorylated proteins such as ovalbumin and egg white riboflavin-binding protein, the phosphorus level of native egg white was 0.6 mg/g. The phosphate group content has grown by more than five times. Moderate microwave treatment alone was shown to boost the phosphorylation levels of egg white proteins, whereas excessive microwave treatment, i.e., 400–600 watts had a detrimental influence on the degree of phosphorylation of the proteins [The degree of phosphorylation of PM-EWP samples under various conditions is shown in i et al. . In the i et al.  discoverp < 0.05). In terms of index d(0.1), the phosphorylation level of 2.5, and 3.5% was more effective in decreasing particle size in lower power microwave samples (p < 0.05). At the same time, the phosphorylation level of 1.5% was more effective (p < 0.05) in decreasing particle size in microwave samples with higher powers, and the particle size of the control samples (P0-M0 and P0-M700) was lower (p < 0.05). In terms of index d(0.5), lower power microwave samples with phosphorylation levels of 3.5 and 2.5% were more effective in lowering particle size (p < 0.05). The particle size of the microwave-assisted phosphorylation samples (p < 0.05) was smaller than that of the control samples (P0-M0 and P0-M700). In terms of index d(0.9), lower power microwave samples with phosphorylation levels of 2.5, and 3.5% were more effective in reducing particle size (p < 0.05). The particle size of the microwave-assisted phosphorylation samples (p < 0.05) was smaller than that of the control sample (P0-M700). Microwave samples with a higher power and phosphorylation levels of 1.5 and 3.5% showed a stronger effect on reducing surface mean diameter (p < 0.05) in terms of index d(3.2) or surface mean diameter. The surface mean diameter of the control samples  was substantially smaller than the surface mean diameter of the microwave-assisted phosphorylation samples (p < 0.05). In terms of index d(4.3) or volume mean diameter, the phosphorylation levels of 2.5 and 3.5% had a greater effect on reducing the volume mean diameter in microwave samples with 200 watts of radiation (p < 0.05). When compared to other PM-EWP samples, the P0-M700, and P0-U0 control samples exhibited the smallest mean volume mean diameter (p < 0.05). It might be because of the improved interaction between protein molecules following treatment with microwaves of 200 and 400 watts, respectively, which caused the protein molecules to gather easily. Arzeni et al. [i et al.  discoveri et al. . Covaleni et al. . This ca−1 examined. The FTIR spectra of microwave-assisted phosphorylation EWPs freeze-dried, as well as control samples, is shown in −1 and 890–900 cm−1, which were attributed to P-O and P=O stretching, respectively [−1) and the amide II band (1480–1575 cm−1) are two absorption bands in the infrared region of proteins that may be used to study secondary structures [−1 and 1397 cm−1, respectively. Microwave-assisted phosphorylation EWPs showed a 2 cm−1 red shift at –NH stretching and a 2 cm−1 blue shift at –CN stretching, compared to the P0-M0 sample, while microwaved-EWP only had a 1 cm−1 blue shift at –CN stretching. In general, microwave-assisted phosphorylation alterations can lead to further structural modifications in proteins [Microwave-assisted phosphorylated samples showed additional adsorption peaks at wave numbers 970–980 cmectively . Tang etectively  found thectively ,33 foundructures . The –NHproteins .In comparison to the control sample (P0-M0), the particle size of microwave-assisted phosphorylated EWPs was decreased . The PM-w/v), water-holding capacity, and foam stability. The binding of negatively charged phosphate groups to the serine, threonine, and tyrosine side chains was discovered by FTIR spectroscopy of the unfolding structure of egg white protein. This binding also caused alterations in the protein’s secondary structure. The phosphorylation and microwave treatments, according to SEM tests, reduced the particle size of the egg white protein powder while increasing the surface area of the protein molecules. According to the findings, microwave-assisted phosphorylation modification followed by freeze drying is a practical approach to enhance the functional qualities of egg white protein. In order to enhance the functional properties of EWP, coupled phosphorylation with non-thermal approaches  is suggested as a direction for future study.In the treatments that used phosphorylation and microwave simultaneously, P1.5-M200 has the highest oil-holding capacity, emulsion stability, and emulsion activity, and P2.5-M200 has the highest foam capacity, P2.5-M400 has the largest particle size, and P3.5-M200 has the highest solution viscosity by 1% (


The complexes have been isolated in a solid state as: fac-[Pt(taci)I3]I·3H2O (1A), fac-[Pt(taci)I3]I (1B), fac-[Pt(taci)I3]I·2DMF (1C), bis-[Pt(taci)2](CO3)2·6H2O (2A) by changing the acidity of the reaction systems, the molar ratios of the reagents and the counterions, and by re-crystallization. The ligand taci is coordinated through the NH2-groups, each molecule occupying three coordination places in the inner coordination sphere of Pt(IV). Monitoring of the hydrolysis processes of 1A and 2A at different acidity showed that while 2A remained stable over the study period, the I−-ions in 1A were successively substituted, with the main product under physiologically mimetic conditions being fac,cis-[Pt(taci)I(OH)2]+ (h2). The antiproliferative tests involved eight cancer cell models, among which chemosensitive (derived from leukemias and solid tumors) and chemoresistant human Acute myeloid leukemia lines , as well as the non-malignant kidney’ cells HEK-293T showed that the complexes 1A and 2A are characterized by a fundamentally different profile of chemosensitivity and spectrum of cytotoxic activity compared to cisplatin. The new Pt(IV) complexes were shown to be more effective in selectively inhibiting the proliferation of human malignant cells compared to cisplatin. Remarkable activity was recorded for 1A, which showed an effect (IC50 = 8.9 ± 2.4) at more than 16-fold lower concentration than cisplatin (IC50 = 144.4 ± 9.8) against the resistant cell line HL-60/CDDP. In parallel, 1A exhibited virtually the same cytotoxic effect against the parental HL-60 cells (IC50 = 9.0 ± 1.2), where cisplatin displays comparable chemosensitivity (IC50 = 8.3 ± 0.8). The determined resistance indices (RI~1) show unequivocally that the resistant lines are sensitive to both compounds tested; therefore, they are capable of overcoming the mechanisms of cisplatin resistance. The structural features of these compounds and their promising pharmacological properties justify their inclusion in the group of “non-classical metal-based antitumor compounds” and are a prerequisite for the admission of alternative mechanisms of action.Metal complexes occupy a special place in the field of treatment and diagnostics. Their main advantages stem from the possibility of fine-tuning their thermodynamic properties and kinetic behavior in the biological milieu by applying different approaches such as properly constructed inner coordination sphere, appropriate choice of ligands, metal oxidation state, redox potential, etc., which are specific to these compounds. Here we discuss the design and synthesis of two octahedral cationic Pt(IV) complexes of the tridentate ligand all- Besidesach)Cl2] . Studiesach)Cl2] ,56,57.The accumulated data on the influence of the structural characteristics of platinum cytostatics on their physicochemical and biological properties, as well as knowledge of possible mechanisms  of theircis-2,4,6-triaminocyclohexane-1,3,5-triol (taci) were obtained, namely with one coordinated taci  and with two coordinated taci molecules , - ions as “leaving” ligands. The carrier ligand, known by the trivial name taci I·3H2O, 1A. Aqueous solutions of K2PtI6  and taci·2H2O  were mixed at a molar ratio Pt:taci = 1.5:1. The mixture was stirred for 3 h while heating at 60 °C. The obtained red crystalline precipitate was filtrated and recrystallized (pH~5). Red crystals of 1A crystallized after a few hours. Elemental analysis, calcd (%) for I4C6H21N3O6PtIV, 1A, (933.91 g·mol−1): C 7.72, H 2.26, N 4.50; found: C 7.61, H 2.22, N 4.44. IR (KBr disks) [cm−1]: 3443, 3358, 3248, 3225, 3171, 2923, 2853, 1621, 1549, 1529, 1514, 1467, 1359, 1332, 1304, 1282, 1208, 1179, 1151, 1072, 1046, 938, 867, 799, 726, 700, 625, 572, 518, 481, 435, 415. 1H-NMR, δ [ppm] (J [Hz]): 3.29(a) ); 3.29(b) ); I(3.29(a):I(3.29(b) = 2 (determined by 195Pt—33.8%); 4.43 ); 13C{1H}-NMR, δ [ppm] (J[Hz]): 48.92 ); 61.41 ); HR-MS [ESI]: [1+] 752.7895 .fac-[Pt(taci)I3]I (1B) and fac-[Pt(taci)I3]I·2DMF (1C) were obtained as follows: for 1B: 0.001 g of 1A were covered with liquid paraffin oil (Nujol) and several days after crystals of 1B were obtained; for 1C: 0.001 g of 1A were dissolved in 2 mL DMF  and red crystals of 1C were grown by slow diffusion of ether into the solution.Crystals suitable for single-crystal X-ray diffraction analysis of bis-[Pt(taci)2](CO3)2·6H2O, 2A. The solution of K2PtI6 obtained by dissolving 0.1699 g (0.16 mmol) in 5 mL distilled water at stirring was mixed with a 5 mL aqueous solution of taci·2H2O  at a molar ratio taci:Pt=2 and the mixture was stirred continuously for an hour at slight heating (t < 40 °C) until the formation of a brown precipitate. The heterogeneous mixture was then heated at 60–70 °C until the brown precipitate became lighter and the pH of the solution reached ~8.5. Then a solution of 0.1 M NaOH was added to pH~9.0–9.5, and the mixture was heated at 60–70 °C for 1-2 h. Yellow crystals suitable for single crystal X-ray diffraction analysis were obtained 1 day after. Elemental analysis, calcd (%) for C14H42N6O18Pt, 2A, (777.51 g.mol−1): C 21.63, H 5.434, N 10.81; found: C 21.77, H 5.055, N 11.24. IR (KBr disks) [cm−1]: 3438, 3155, 3073, 3015, 2926, 2844, 2705, 2657, 1606, 1572, 1473, 1410, 1394, 1372, 1347, 1240, 1172, 1073, 976, 966, 887, 821, 599, 539, 473. 1H-NMR, δ [ppm] (J[Hz]): 3.04(a) ); 3.04(b) ); I(3.04(a):I(3.04(b) = 2 ; 4.33 ); 13C{1H}-NMR, (δ [ppm] (J[Hz]): 50.50 ); 63.47 ). HR-MS [ESI]: [(2-3H)+] 546.1643 .1H NMR experiments were conducted as samples of 1A (1.1 × 10−3 mmol) and 2A (1.3 × 10−3 mmol) were dissolved in 1 mL D2O solutions. Mass spectra were recorded in water solutions (1 mg/L) at room temperature. The proper pH values were achieved by adding 0.01 M NaOH (NaOD for NMR). The experiments were also conducted in commercially available PBS . The reactions were followed immediately after sample dissolving and every 24 h in a 72 h period. In order to closely simulate the human body’s cellular conditions, the solution behavior of 1A and 2A in PBS (pH = 7.4) were also monitored at 37 °C by 1H-NMR.The investigations on the stability of the complexes in solution were performed by means of NMR spectroscopy and mass spectrometry. The 1A exhibits substitutional disorders over two water molecules (O4S and O5S), which were refined with occupancy of 0.33 and 0.50, respectively. The delocalized solvent’s molecules in 1A were handled with the SQUEEZE function of PLATON software -2,5-diphenyl-2H-tetrazolium bromide (MTT)-dye reduction assay as described by Mosmann with minor modifications + of the platinum salt with donor atoms from taci (3(taci)]I·3H2O, 1A. The great stability and low reactivity of Pt-I bonds is the reason for the coordination of only one molecule of the tridentate ligand taci through substitution, most probably of the three water molecules from the inner coordination sphere of Pt(IV). The charge of the obtained cationic complex [PtI3(taci)]+, 1 is neutralized by an iodide liberated during the hydrolysis of the starting platinum salt.An aqueous solution of Ke stages  though srom taci . The int2](CO3)2·6H2O, 2A. It appears that in order to coordinate the second taci molecule, it is necessary to substitute the three iodides from the inner coordination sphere of complex 1 with hydroxido ligands  ligands  present 2 in a solid state as 2A is the presence of CO32- ions in the reaction system. As an alicyclic primary triamine, in an aqueous medium at pH > 6.0, the ligand taci interacts with CO2 I, 1B, where the water molecules have left the crystal lattice of 1A. The compound 1B was obtained by crystal-to-crystal transformation from 1A under liquid paraffin oil. Complex 1 was also isolated as crystals with two molecules of DMF as fac-[PtI3(taci)]I·2DMF, 1C after re-crystallization of 1A in DMF.A peculiarity in obtaining the complex with CO2  from the of taci  by adjusfac-[PtI3(taci)]+, 1 and bis-Pt(taci)2]4+, 2 with the atom numbering are shown in Projections of the complex cations: 1A crystallizes in a monoclinic crystal system, space group P21/c (fac-[PtI3(taci)]+ (noted as 1A-A and 1A-B)), two I- anions, and a set of co-crystallized water molecules. The ligand taci is coordinated as a neutral molecule, and the formation of three identical six-membered chelate rings as Pt-N-C-C-C-N  in the complex cation  and Pt-N(amine) (2.107 Å) bonds (fac-derivative of the terdentate diethylenetriamine ligand with three iodine atoms around Pt(IV) + and an I− anion. In 1B, the difference between bond lengths Pt-N (Δ 0.0067 Å) and Pt-I (Δ 0.0043 Å) is smaller than this in 1A, but the deviation of the bond angles from 90° is up to 5°  bonds  for the d Pt(IV) . In the  of 100% . The comce group  and consup to 5° . Hence tive cell . In thisolecules . The cry2A (ditaciplatin) crystalizes in monoclinic C2/c crystal system (2-groups which occupy the six available coordination sites in the inner coordination sphere of platinum(IV)  and are shorter than the third one (2.078 Å). Since the opposite Pt-N bonds from the two coordinated taci molecules are identical and considering the negligible deviation of the angles up to 1.5°, it is accepted that the complex cation in 2A adopts a slightly elongated octahedral geometry. The asymmetric unit of 2A contains half of the complex molecule, half of each of the two counter anions, and three water molecules. In the asymmetric unit platinum center is closely surrounded by two water molecules (O1S and O3S) and one carbonate (C8) by H-bonds: O1S-H1SA-O6, O3S-H3SB-O4, O2-H2-O10, O2-H2-O8, N3-H3A-O8, N3-H3A-O9). The second carbonate (C7) and the third water (O2S) are also engaged with H-bonds: O2S-H2SA-O8 and O2S-H2SA-O9  b, and th-H2SA-O9 b. Thus, -H2SA-O9 . The 3D -H2SA-O9 . The two1A and 2A was proved based on their spectroscopic characteristics. The mode of taci coordination was investigated by solid-state FTIR spectroscopy , and these to the out-of-plane (wagging) vibrations are shifted to higher frequencies, and the bands assigned to the NH2-twisting vibration are shifted to lower frequencies. The observed shifts of the bands originating from the NH2-groups in the spectra of the complexes prove unambiguously the symmetrical coordination of the ligand taci through the three NH2 groups. The facial coordination of the three NH2-groups and the three I− in 1A was also supported by the sets of three bands for stretching Pt-N vibrations in the region 520–430 cm−1 and for Pt-I vibrations in the region 310–250 cm−1. The equivalent coordination of six NH2 in an octahedral environment from two taci ligands in 2A was supported by only one observed band for Pt-N stretching at 539 cm−1. The presence of planar CO32− (D3h) in the outer coordination sphere of 2A was proved by bands for the four normal modes of vibration . The individual C-atoms have separated signals because of the lower molecular symmetry in the crystal structure. The signals of carbons attached to OH are narrower and well separated. Conversely, the signals of carbons attached to the NH2- groups are broadened and thus confirmed again their close location to platinum in the molecules. The presence of two carbonates in 2A was confirmed from downfield signals at ~168 ppm and 167 ppm. A signal of the coordinated platinum(IV) at −746 ppm was also acquired in the 195Pt CP-MAS of 2A. Overall, the signals in the solid-state spectra of 1A are broader, and it is assumed that this is a consequence of the effect of coordinated in the inner coordination sphere of three I- ligands together with the three NH2 groups.The measured solid-state NMR spectra were a very useful tool for the structural description of the powder sample of m for 1A  and −14.r 1A (2A ) comparef 179 Hz . The obsompounds , two set1A and 2A (10−3 mol/L) are 5.5–6.0 and 9.0–9.5, respectively. The HR-ESI(+)-MS spectra recorded of the fresh water solutions of the compounds (~10−6 mol/L) display the specific platinum isotopic pattern. The main signals of the corresponding spectra were detected at m/z: 752.7895 for 1A and 546.1643 for 2A, and they are consistent with a 1:1 complex with a composition [Pt(taci)I3]+, 1 and 1:2 complex—[Pt(taci)2-3H]+, 2 , as one of them belongs to the ring hydrogens attached to C-N and the other—to those attached to C-O. The position of the two signals depends on the acidity of the systems +(h2). The architecture of the inner coordination sphere of h2 can be seen as favorable for a mechanism based on covalent interaction with biomolecules by substituting the two cis-coordinated OH-groups. The unsubstituted I--ligand by means of a cis-effect can further favor the processes of substitution, for example, with the nucleic bases, and the side OH-groups of taci can stabilize additionally the adducts formed in the biological environment through H-bonding. It appears that the reduction processes to Pt(II)-active species will not be decisive due to the absence of steric hindrance caused by the bulky axially positioned ligands or the absence of two trans-disposed ligands in the inner coordination sphere with high electronegativity. The proposed mechanism seems unacceptable for 2A since there are no factors provoking fast hydrolysis processes, and its less pronounced cytotoxicity can be explained by a direct redox reaction with biomolecules, here including those that are hallmarks of cancer. It should be emphasized that mechanisms based on direct oxidative damage to DNA or other biomolecules could be possible for both complexes. A distinctive feature of the biological behavior of the two investigated compounds is that each of them shows approximately the same cytotoxic effects on the resistant cell lines included in the study and the cells of the parental lines, as a quantitative measure of these effects is the values of the resistance indexes that were determined close to 1. In addition, both test compounds were shown to be much more effective than cisplatin in selectively inhibiting cancer proliferation, exhibiting toxicity at times higher concentrations against the renal non-malignant cells HEK-293T. Therefore, the new compounds exhibit a fundamentally different mechanism of cytotoxicity, and without a doubt, they possess the potential to overcome the cisplatin cellular self-defense mechanism. These results, together with ongoing preliminary studies that show a cell death pathway distinct from cisplatin, demonstrate the potential of the new Pt(IV) complexes in antitumor drug development.The biological studies were performed with the complexes taciplatin I3]I·3H2O and bis-[Pt(taci)2](CO3)2·6H2O have been synthesized and named as taciplatin and ditaciplatin. The ligand in the complexes is symmetrically coordinated through the NH2 groups. The solid-state structure of the compounds was studied using X-ray diffraction, solid-state NMR, and FTIR experiments, and the agreement between single-crystal and powder samples was demonstrated. The new platinum complexes exhibited favorable physicochemical properties in water solutions, and under physiologically relevant conditions, the 1:1 complex, taciplatin, revealed relatively fast hydrolysis with substitution of the inner-sphere I- ligands. Its main hydrolysis product on the fifth day under physiological conditions is fac,cis-[Pt(taci)I(OH)2]+ with two substituted I-. In this species, the two cis-located OH-ligands occupying an equatorial position provide active centers for further attacks in biological systems. The antiproliferative tests involving eight chemosensitive and chemoresistant cell lines demonstrated the concentration-dependent activity of the new compounds, with the activity of taciplatin being superior. The difference in antineoplastic activity between taciplatin and ditaciplatin appears to be due to the slower hydrolysis kinetics of ditaciplatin, which results from the structure of its inner coordination sphere. More importantly, the two compounds displayed higher activity against the resistant cell lines included in this study, as remarkable activity was reported for taciplatin, which exhibited the effect at more than 16-fold lower concentrations than that of cisplatin against the HL-60/CDDP cell line. The data analysis of the biological studies unambiguously highlights the differences of the presented “applicants” for antineoplastic agents with respect to cisplatin and, in particular, their potential in the treatment of resistant cell lines. Moreover, their specific structural features based on octahedral platinum(IV) complexes of the ligand with favorable properties give reasons for more in-dept biological experiments. In this regard, taciplatin deserves special attention. Its octahedral structure constructed by three N-donors of a tridentate ligand and three I- ligands give advantages in respect to control the kinetic behavior, the strength of the M-L bonds in the biological milieu, as well as for application in photodynamic therapy. The proposed new structure of the inner coordination sphere that the new compounds show, together with their promising pharmacological properties, warrants their inclusion as new proposals in the group of platinum-based non-classical antitumor drugs.In search of a new formulation for antineoplastic drug candidates, two octahedral Pt(IV) complexes of the tridentate ligand all-


These results highlight a new kind of uncertainty that needs to be considered when incorporating new observational data into model forcing while also raising questions about the role of BB emissions on the observed Arctic sea ice loss.The mechanisms underlying decadal variability in Arctic sea ice remain actively debated. Here, we show that variability in boreal biomass burning (BB) emissions strongly influences simulated Arctic sea ice on multidecadal time scales. In particular, we find that a strong acceleration in sea ice decline in the early 21st century in the Community Earth System Model version 2 (CESM2) is related to increased variability in prescribed BB emissions in the sixth phase of the Coupled Model Intercomparison Project (CMIP6) through summertime aerosol-cloud interactions. Furthermore, we find that more than half of the reported improvement in sea ice sensitivity to CO The sensitivity of sea ice to fire emissions highlights climate model uncertainty related to the accuracy of prescribed forcings. Arctic sea ice has experienced marked reductions in extent, thickness, and volume in recent decades, making it one of the most striking manifestations of anthropogenic climate change. Sea ice loss has been observed in all months of the year  historical simulations  . Previous studies suggest that the aerosol forcing of CMIP5 simulations might have been too weak in recent decades . AlthougTo isolate the impact of the increased BB variability over the GFED era on Arctic sea ice, we conducted sensitivity ensemble simulations (referred to as CESM2-BB hereafter) in which the interannual variability in BB emissions from 1997 to 2014 between 40° and 70°N is removed but the integrated amount of emissions over that same period is retained . BecauseThe sensitivity experiments show that the warming of the Arctic (70° to 90°N) over the GFED period is more pronounced in the CESM2-LE compared to the CESM2-BB , with thAround year 2010, the trend in Arctic warming and sea ice decline starts to lessen in the CESM2-LE , slightlThe impact of BB emissions on Arctic climate can be explained by aerosol-cloud interactions . Freshly2 emissions  Community for CMIP6 in general . From the 12 additional CMIP6 models assessed here , 4  show an accelerated ensemble mean sea ice loss over the GFED period, although none of them is as large as the CESM2. This indicates that the impact of BB emissions is likely not just limited to the CESM2 but may affect other CMIP6 models as well, in agreement with results from a companion paper that finds increased surface downwelling shortwave radiation during the GFED period in several CMIP6 models in addition to the CESM2  Sea Ice Index version 3  the presence of sporadic large CO2 uptake over land  in which we averaged BB emissions from 1997 to 2014, computed on a monthly basis, such that BB emissions have a fixed annual cycle while keeping the same integrated amount of emissions over that same period. This approach is identical in nature to what was used in CMIP5 , which also uses homogenized BB emissions to avoid the increase in BB variability over the GFED era  are as follows: ACCESS-CM2 . Using aThe CMIP6 models are separated into a sensitive and not sensitive category based on whether they exhibit a similar sensitivity to the increased variability in BB emissions as the CESM2-LE (figs. S6 and S7). First, we calculate 20-year linear trends in September sea ice area for each model and compare the slope of the 20-year linear trends between the reference period of end years 1978–1990 and the acceleration period of end years 1997–2009. Note that we chose the last year of the acceleration period to be 2009 instead of the last year of the GFED era  based on when the CESM2-LE and CESM2-WACCM reach their maximum negative September sea ice area trend (see fig. S7). For a model to be characterized as sensitive, the slope of sea ice area trends over the acceleration period needs to be at least two times larger  than the slope of sea ice area trends over the reference period. This criteria is defined on the basis of the relative increase in sea ice trend for each model to account for the different magnitudes of sea ice loss across all CMIP6 models (fig. S7). We decided to choose two periods of the same length and to exclude the years 1991–1996 from the reference period because of the Mount Pinatubo volcanic eruption in 1991 and the global cooling that followed for a few years, which resulted in a peak increase in Arctic sea ice extent about a year and a half after the eruption in some models (


Neurospora circadian oscillator, the negative element, FREQUENCY (FRQ), inhibits its own expression by promoting phosphorylation of its heterodimeric transcriptional activators, White Collar-1 (WC-1) and WC-2. FRQ itself also undergoes extensive time-of-day-specific phosphorylation with over 100 phosphosites previously documented. Although disrupting individual or certain clusters of phosphorylation sites has been shown to alter circadian period lengths to some extent, it is still elusive how all the phosphorylations on FRQ control its activity. In this study, we systematically investigated the role in period determination of all 110 reported FRQ phosphorylation sites, using mutagenesis and luciferase reporter assays. Surprisingly, robust FRQ phosphorylation is still detected even when 84 phosphosites were eliminated altogether; further mutating another 26 phosphoresidues completely abolished FRQ phosphorylation. To identify phosphoresidue(s) on FRQ impacting circadian period length, a series of clustered frq phosphomutants covering all the 110 phosphosites were generated and examined for period changes. When phosphosites in the N-terminal and middle regions of FRQ were eliminated, longer periods were typically seen while removal of phosphorylation in the C-terminal tail resulted in extremely short periods, among the shortest reported. Interestingly, abolishing the 11 phosphosites in the C-terminal tail of FRQ not only results in an extremely short period, but also impacts temperature compensation (TC), yielding an overcompensated circadian oscillator. In addition, the few phosphosites in the middle of FRQ are also found to be crucial for TC. When different groups of FRQ phosphomutations were combined intramolecularly, expected additive effects were generally observed except for one novel case of intramolecular epistasis, where arrhythmicity resulting from one cluster of phosphorylation site mutants was restored by eliminating phosphorylation at another group of sites.In the negative feedback loop driving the  Neurospora, the WCC transcription activator drives the expression of FRQ, which complexes with FRH and CK1 to repress the DNA-binding activity of WCC by promoting phosphorylation at a group of residues of WCC. The phosphorylation status of FRQ determines the circadian period length, acting independently of effects of phosphorylation on FRQ half-life. Reflecting this dominant role of phosphorylation, FRQ is subject to substantial phosphorylation at over 100 sites in a time-of-day-specific manner. However, how this plethora of phosphoevents on FRQ controls its activity in a circadian cycle is still elusive, and prior work had shown limited effects of individual phosphosite point mutants. In this study, a series of frq mutants targeting multisite phosphorylation within domains of FRQ were generated and analyzed in order to define their roles in period determination. A clear pattern of period-altering effects was observed in these frq mutants; certain mutants display strong temperature compensation phenotypes, and interestingly, a novel epistatic relationship on rhythmicity between phosphogroups emerged.Circadian rhythms, found in most eukaryotes, are based on cell-autonomous, auto-regulatory feedback loops in which negative elements feed back to depress their own expression by repressing the positive elements that drive their synthesis. In Living organisms on earth are persistently under the influence of external light/dark cycles. To anticipate and, more importantly, better utilize these environmental cues, most organisms have evolved an internal cellular oscillator, the circadian clock, that integrates daily signals, such as light, temperature, and chemicals, to metabolism  bring about repression to their transcriptional activators, WC-1 and WC-2 in Neurospora, Clock (Clk) and Cycle (Cyc) in Drosophila, and BMAL1/Circadian Locomotor Output Cycles Kaput (CLOCK) in mammals, to terminate their own expression, thereby closing the circadian feedback loop , a heterodimer comprised of WC-1 and WC-2, serves as the transcriptional activator for the pacemaker gene frequency (frq) by binding to one of the two DNA elements in the frq promoter: the Clock box (C-box) in the dark  in the light . Four primer sets were used as flanks to facilitate homologous recombination in a yeast strain (FY834) by which point mutations of frq were introduced from PCR primers. To introduce mutations to aa 1–214 of FRQ, two PCR reactions were performed: one with a forward primer “frq segment 1F” (5′-GAACCAGAACGTAGCAGTGTG-3′) and a reverse primer “#pA R” bearing a point mutation(s) to FRQ and the other using a forward primer “#pA F” which is reverse and complementary to “#pA R” and a reverse primer “frq segment 1R” (5′-GACGATGACGACGAATCGTG-3′), and then the two PCR products were co-transformed into yeast along with pCB05  and “frq segment 2R”  and “frq segment 3R” (5′-CAGCACATGT TCAACTTCAT CAC-3′) were designed for pCB05 digested with NruI and FseI (FRQ aa 438–675), and “frq segment 4F” (5′-CACCGATCTTTCAGGAGACCCTG-3′) and “frq segment 4R” (5′-CACTCAGGTC TCAATGGTGA TG-3′) work for pCB05 digested with FseI and MluI (FRQ aa 676–989). If multiple phosphosites span two or more PCR segments mentioned above, corresponding restriction enzymes and primers encompassing the region were chosen and combined for recombination in yeast. All mutations were verified by cycle sequencing at the Dartmouth Core facility. The open reading frame of frq bearing 84 phosphomutations (84Afrq) from , 5 μl of 10 mM MnCl2, and 2 μl of lambda protein phosphatase  were added to the washed FRQ-coupled V5 resin. The mixture was incubated at 30°C for 30 minutes, and then 50 µl of 5 × SDS sample buffer was added and heated at 99°C for 5 minutes  from 2 mg of centrifugation-cleared lysate, FRQ-bound V5 agarose was thoroughly washed twice using the protein extraction buffer, and all supernatant was carefully removed by pipetting. To make a total reaction volume of 52 µl, 40 μl of HFor WB, equal amounts (15 μg) of cleared protein lysate were loaded per lane in an SDS-PAGE gel. FRQ, FRH, WC-1, and WC-2 antibodies were previously described  that contains the frq-C-box fused to the codon-optimized firefly luciferase gene  at the his-3 locus.Luciferase assays were performed as previously described (frq mutants (replacing Ser/Thr with Ala) covering all the 110 phosphosites in a group manner  1 to 682 of FRQ were mutated to Ala altogether, and 27pAfrq bears Ala mutations to the 27 phosphosites in aa 683–989 of FRQ. Consistent with the arrhythmicity observed in 84pAfrq and 110pAfrq  (Δ899–989frq (18.7 hours)  (C23Afrq (18.97 hours) , while 708–989pAfrq displays a decreased period length (14.9 hours) (27pAfrq (1–259pAfrq bears mutations in and near to the coiled-coil domain that is required for FRQ to interact with itself and other core clock components (115–193pAfrq and 194–220pAfrq). Phosphorylation surrounding the coiled coil (CC) and NLS was eliminated in 115–193pAfrq and 194–220pAfrq, respectively, which showed periods of 20.7 and 26 hours, respectively .To directly examine the overall effect of FRQ phosphorylation on period length, we first made two mutants, frq110pA , frq57pA.1 hours , shorterno acids . In frq19 hours) , consist shows a decreased period  (bdras-1 (clock WT), had normal temperature compensation, and the positive control, bdras-1, prd-3  . The negd, prd-3 . Compared, prd-3 . Statistd, prd-3  indicatefrq mutant strains bearing fewer, more select mutations were generated to these and their neighboring regions   but is robustly rhythmic, albeit with a longer period length  may be due to reduced CK1 interaction, consistent with an observation that the period length is determined by FRQ-CK1 interaction , S541A, S545Afrq (+3 hours), and S632A, S643Afrq (+3 hours)  were combined intramolecularly; examples include .2 hours , exactlyfrq mutant—S72A, S73A, S76A, S238A, S240A, S390A, S392A, S394A, S538A, S540A, S541A, S545A, S548A, S551A, S554A, S632A, S634Afrq—which, unexpectedly, displays a loss of rhythmicity  (S900Afrq (∼18 hours)  or Glu (E) are a widely used strategy to simulate phosphorylation by constitutively introducing a negative charge into a domain. In T period ; similarctively)  as frqS9frq phosphomutants covering all 110 phosphosites, and detailed mutagenetic analyses have allowed circadian roles of these site assigned to different domains of FRQ   results in a short period  become hyperphosphorylated later, peaking at CT12, suggesting that these phosphorylations may function in inhibiting FRQ activity. Consistent with these, 1–114pAfrq and 537–570pAfrq develop long periods of 26.7 and 34.8 hours, respectively . Due to scarcity of purified FRQ for in vitro studies and potential ionization issues of peptides bearing multisite phosphorylations in mass spectrometry, whether phosphorylation of FRQ at many sites changes in concert in a circadian cycle is still largely unknown, which restricts our understanding of the role of time-specific phosphoclusters on FRQ.FRQ phosphorylation dynamics have been investigated by quantitative mass spectrometric analyses including stable isotope labeling by amino acids in cell culture (SILAC) (t period , suggestectively  and 6b. Drosophila and PERs/CRYs in mammals act as the negative elements in the negative feedback loop by inhibiting Clk/Cyc and CLOCK/BMAL1 activities, respectively, terminating their own expression and thereby closing the circadian negative feedback loop. Similar to FRQ and WCC in Neurospora, PER/TIM and PERs/CRYs also undergo extensive phosphorylation, and phosphorylation of PER/TIM and PERs/CRYs has been shown to be a critical mechanism in controlling both the fly and mammalian clocks (Results in this work may inform understanding of mammalian and insect clocks, many facets of which are also built on time-specific multisite phosphorylation events to the key components (reviewed in Lastly, we noted that in the case of FRQ phosphorylation at S900, an aspartic acid or glutamic acid substitution could not faithfully mimic the effect of phosphorylation ; also, ijkac334_Supplementary_DataClick here for additional data file.


Sea ice in the marginal ice zone (MIZ) consists of relatively small floes with a wide size span. In response to oceanic and atmospheric forcing, it behaves as an approximately two-dimensional, highly polydisperse granular material. The established viscous-plastic rheologies used in continuum sea ice models are not suitable for the MIZ; the collisional rheology, in which sea ice is treated as a granular gas, captures only one aspect of the granular behaviour, typical for a narrow range of conditions when dynamics is dominated by binary floe collisions. This paper reviews rheology models and concepts from research on granular materials relevant for MIZ dynamics (average stress as a result of ‘microscopic’ interactions of grains; This article is part of the theme issue ‘Theory, modelling and observations of marginal ice zone dynamics: multidisciplinary perspectives and outlooks’. Ice fragmentation is a result of many dynamic processes, including breaking by waves, shear deformation and floe collisions, combined with thermodynamic influences, e.g. upwelling of relatively warm water masses at the ice edge. The resulting floe size distributions (FSDs) in the MIZ often are scale-invariant in the size range of relatively small floes and have exponential tails related to the existence of the upper size limit, i.e. they are well described by a tapered power law . The relIn parallel with significant progress in research on sea ice dynamics at the floe scale, briefly sketched above, substantial developments have taken place in research on granular rheology, including that of dry granular materials and suspensions. One of the main goals of this study is to provide a concise—and, by necessity, rather selective—review of ideas that seem relevant for sea ice and that might inspire further research on MIZ dynamics and, in a further perspective, lead to the development of a MIZ rheology model capturing the whole range of behaviours, from quasi-static, dense flow in the inner MIZ to dilute, inertial flow close to the ice edge. To this end, after introducing basic definitions related to the rheology of viscous fluids in §2a, the assumptions and properties of several variants of the so-called . 2Anticipating the application of concepts presented in this section to continuum modelling of sea ice, in which the momentum and constitutive equations are formulated in two dimensions (integrated over ice thickness), the description and formulae in §2a–c below are given for two-dimensional materials as well. Full three-dimensional versions can be found in the cited papers. Cartesian coordinates are used in the horizontal plane, with axes  (a)Continuum models of granular materials, analogously to models of viscous fluids, are based on the conservation equations for mass and momentum, the general form of which ise, e.g., ). Apart s fluids 2.4σ^ic=rheology ,28 and t (b)Based on a dimensional analysis of the governing equations describing a simple-shear flow (er (e.g. )2.5I=ρpd ϕ=ϕ(I) –32. The given by  with σ^iy (σ^ijc 2.6σ^ijcre (e.g. )2.7μ(I)inations , the μ o,35 oε˙PSFrom the point of view of the potential application of a ssion in ).It is also worth noting that if e, as in , I can bned with , the preher with , makes tFinally, the inertial number squared, contacts ,40. Thus (c)DEM methods are an invaluable source of information on granular flows as they allow to relate processes acting at different scales, from grain-level interactions, through contact/force chains and grain clusters, to bulk material properties relevant for continuum models. Obtaining that multi-scale information from observational data is an unattainable goal. Obviously, in order for DEM results to be useful, the relevant bulk variables must be computed from information available at the grain level in a proper way. Considering the wide popularity of DEM methods, it might seem surprising that misconceptions regarding the computation of volume-averaged stress are quite common, and several papers are devoted to alternative definitions of that quantity and their interpretation. It is therefore useful to provide here a summary of that computation and the relevant concepts.a), In the momentum equations , there ais e.g. ,42)2.8σ2.8σV th side of , which ains . A fund the MIZ , the modty (e.g. ). Wherea used in ,49 and rA substantial development of the collisional rheology was relaxing the simple-shear assumption of . Based oons (see  for deta. 4 (a)As mentioned in the introduction, a DEM model is used in this study to illustrate selected aspects of the discussed processes. To this end, the DESIgn model  is run iribed in 4.1p(r)∼sed : the lower, ‘frozen’ boundary for During the preparation phase of each run group, the floes are placed randomly, without overlaps, within a square domain with surface area  regions b: the loloe (see ). The bogiven by  and the l number  becomesfined in . Notablyfined in  reduces All simulations are repeated in two versions: with boundary forcing only (version A); and with ice–water drag, assuming ocean at rest (version B). In version B, the  (b)a shows the shear stress a; notably, some rearrangements are present even at b).The model version A, without any forces from the ocean or atmosphere, represents a dry granular material . The results of that version are very useful as a reference case, for which the role of polydispersity in the material’s response to shear deformation can be analysed in detail. Figure 2b), are closely related to the presence of the largest floes in the ensemble. If all floes are small, a,b). The presence of floes with diameters comparable with the size of the domain disrupts the uniform character of the flow. As the floes move as rigid bodies, velocity a)—the net flow becomes sensitive not only to the sizes, but also to the relative positions of the small number of the largest floes and, moreover, it has a fully two-dimensional character, i.e. due to small floes flowing around the edges of the large ones, the variance of b). The net, macroscopic result is a larger stiffness of the ice described above, i.e. lower strain rates for a given shear stress or, vice versa, higher stress under a given strain rate. Importantly as well, large size span of floes leads to strong temporal fluctuations of stress, related to phases of a relatively smooth flow separated by events of sudden blocking (not shown).Those FSD-related effects, including the decrease of easing α b, are clirection a,b. The a), the resulting combinations of a) which can be well approximated with formula (a that strongly deviate from the b), although, as expected, the range of obtained shear rates and thus inertial numbers is narrower . A less trivial result is that transient jamming at high b than in figure 4a).In spite of the very wide range of nsidered a, the reed curve a which c formula . AlthougFinally, in both versions and in all cases considered the ice concentration sumed in , the relsumed in . As the  formula  should b. 5Granular materials are mesoscopic—in their dynamics, the microscopic (grain-level) temporal and spatial scales are not well separated from the macroscopic scales. The lack of scale separation is related not only to the fairly large ratio of grain size to the dimensions of systems typically analysed in practical problems , but, most importantly, to the nature of interactions between grains ,58,59. TNot surprisingly, all those properties lead to difficulties in continuum modelling, for which separation of scales is a prerequisite. Yet another difficulty—contrary to those mentioned above related specifically to sea ice—are increasing spatial resolutions of continuum sea ice models, especially those operated at regional and local scales. Although, indisputably, that development is very positive, the drawback is that even in the MIZ, where ice floes are relatively small, continuum rheologies have to deal with floe sizes comparable with mesh sizes of the models. The results presented in this paper suggest that, on the one hand, the average stress can be reliably computed and the strain–stress relationships remain relatively stable up to high floe diameter to mesh size ratios, but, on the other hand, the information on FSD properties is crucial for that computation.Another information that might play a role, not analysed in this study  are floe shapes. In the simulations in §4, the floes were disc-shaped and no rolling resistance was included in the contact model. This might explain the rather low values of friction coefficients obtained . The sparse evidence available, based on recent ice tank experiments and DEM simulations, indicates that floe shapes influence the stability of force networks and therefore stress in the ice, as well as ice-induced loads on structures (e.g. ). At theApart from the influence of FSDs on stress in the ice, a very robust feature clearly seen in the results presented in this paper  is the dependence of the friction coefficient ld curve ,28. Thusmulation , μ=4π/3( Feltham  v′=2T an