However, the complex relationship among halidesubstitution, structural disorder, and lithium distribution is notfully understood, impeding optimal material design. In this study,we investigate the effect of bromide substitution on lithium argyrodite and engineer structural disorder by changingthe synthesis protocol. We reveal the correlation between the lithiumsubstructure and ionic transport using neutron diffraction, solid-statenuclear magnetic resonance (NMR) spectroscopy, and electrochemicalimpedance spectroscopy. We find that a higher ionic conductivity iscorrelated with a lower average negative charge on the 4d site, located in the center of the Li+ “cage”,as a result of the partial replacement of S2– byBr–. This leads to weaker interactions within theLi+ “cage”, promoting Li-ion diffusivityacross the unit cell. We also identify an additional T4 Li+ site, which enables an alternative jump route (T5–T4–T5)with a lower migration energy barrier. The resulting expansion ofthe Li+ cages and increased connections between cages leadto a maximum ionic conductivity of 8.55 mS/cm for quenched Li5.5PS4.5Br1.5 having the highest degreeof structural disorder, an 11-fold improvement compared to slow-cooledLi6PS5Br having the lowest degree of structuraldisorder. Thereby, this work advances the understanding of the structure–transportcorrelations in lithium argyrodites, specifically how structural disorderand halide substitution impact the lithium substructure and transportproperties and how this can be realized effectively through the synthesismethod and tuning of the composition.Lithium argyroditesuperionic conductors have recently gained significantattention as potential solid electrolytes for all-solid-state batteriesbecause of their high ionic conductivity and ease of processing. Promisingaspects of these materials are the ability to introduce halides (Li Further,improving conductivity and developing effective synthesis routes demanda better understanding of the conduction mechanism and its correlationto the structure.Lithium-ion batteries are the leadingbattery technology used inportable electronics and electric vehicles, demonstrating constantenhancement/improvement in electrochemical performance resulting fromboth materials and engineering development.266PS5Hal) argyroditein the ordered state having the cubic F4̅3m space group. In the face-centered cubic lattice, halideions occupy the tetrahedral Wyckoff 4a sites andS2– not bonded to P occupies half of the tetrahedralsites (Wyckoff 4d). The argyrodite cubic phase anionframework forms 136 interstitial tetrahedral voids per unit cell availablefor cation occupancy.29 Four voids are occupied by P5+ cations on the Wyckoff 4b site, forming PS4–3 tetrahedra, together with S on theWyckoff 16e site. The remaining 132 voids can beoccupied by the lithium sites and categorized according to how manyS ions (16e) are shared  withthe PS4–3 tetrahedra.30 Thus, the tetrahedral site can be split into five distinct tetrahedraltypes (types 1–5), as shown in 32 The type 1 (T1) and type 2 (T2)sites share faces and edges with PS4–3. The type 3 (T3) and type 4 (T4) tetrahedral sites share 4and 3 corners with PS4–3, respectively.33 The type 5 (T5) sites share 2 corners with PS43– and the type 5a (T5a) sites lie in betweenthe faces of 2 neighboring T5 sites in a trigonal bipyramidal environment11 because of their close proximity, as shown in d site), as shown in + networkthroughout the structure.The halide-based argyrodite family is consideredone of the mostpromising classes of solid electrolytes exhibiting very high ionicconductivities at room temperature.35 proposed threedifferentjump processes for lithium-ion conduction pathways: intracage anddoublet jumps (within the cage) and intercage jump (between the cages).35 In these DFT simulations, T5 and T5a Li siteswere considered, whereas a recent study has suggested additional lithiumsites to be involved that impact the diffusion pathway and mechanism.37 Minafra et al. recently reinvestigated the lithium argyrodite substructurefor Li6PS5Hal , revealing additionalT2 lithium occupancies and suggesting new lithium conduction pathways.37 In other sulfide argyrodite compositions, apartial occupancy of additional  lithium sites wasdiscovered.40 For instance,the partial substitution of Ge4+ in the P5+ sitein Lix6+Px1–GexS5I found a partialoccupancy of T2 and T4 sites,41 which providesshorter and favorable paths for Li+ diffusion, therebyincreasing the ionic conductivity from 10–3 to ∼10mS/cm.41 Another argyrodite series, theSn-substituted thioantimonate argyrodites, exhibited T2 and T3 siteoccupancies, and Li6.15Al0.15Si1.35S5.4O0.642 exhibitedT4 site occupancies.42De Klerk et al.d site).43 The iodide version represents the ordered arrangement where the4a site is fully occupied by I– (I– (Wyckoff 4a) = 1), while the 4d site is exclusively occupiedby S2–, a result of the large difference in ionicradii. In this case, poor ion diffusion between the Li-ion cages presentsthe rate-limiting step for long-range diffusion.35 Following the decreasing difference in ionic radii, thesite disorder increases for bromide, followed by the chloride versionhaving the highest site disorder of S–2/Hal– on the 4d site (Br– (4d) = 20%, Cl– (4d) = 60%).43 DFT simulations predict that the maximum conductivitymay be achieved with around 75% site disorder, where the jump ratefrequency of the three jump processes, doublet, intra-, and intercage,is similar.35 Halide substitution is alsoshown to increase conductivity. Adeli et al.26 investigated the higher chloride substitution in Lix6–PSx5–Clx1+ (x ≤ 0.5) with a conductivityof 9 mS/cm achieved at x = 0.5. In addition to differenthalides, Yu et al.44 reported an ionicconductivity of 4.13 mS/cm for the higher bromide-substituted Lix6–PSx5–Brx1+ (x ≤0.5).44 Despite these significant advances,the relationships between long-range diffusion and structure, especiallywith regard to sublattice disorder, are still not well understood.Anion sitedisorder refers to the random arrangement of variousanions within the same crystallographic site  on lithium argyrodite and engineer structuraldisorder by changing the synthesis protocol. First, an argyroditeprecursor was prepared via mechanical milling, followed by heat treatmentat a particular temperature to obtain crystalline argyrodite. Twodifferent cooling methods were employed to influence the extent ofstructural disorder. Rietveld refinement against the neutron diffractionpatterns was performed, demonstrating that this preparation methodinfluences the structural disorder. We found that higher ionic conductivityis correlated with a less negative charge on the 4d site as replacing S2– with Br– leads to a lowered average charge on the 4d siteand weaker interactions within the Li+ “cage”,promoting a diffusion pathway for Li+ ions across the unitcell. We also identified an additional T4 Li+ site in bromide-basedargyrodite, which enables an alternative jump route (T5–T4–T5)with a lower migration energy barrier. The resulting expansion ofthe Li+ cages and the increased connections between thecages lead to a maximum ionic conductivity of 8.55 mS/cm. Overall,this work provides a deeper understanding to further improve the ionicconductivity of lithium argyrodites and other solid electrolytes.In this study, we investigate the effect of halide substitution(Lix6–PSx5–Brx1+ syntheses were performed under an argon atmosphereto prevent the contamination of oxygen (O2 < 2 ppm)and water (H2O < 1 ppm). Lix6–PSx5–Brx1+ was synthesized using mechanochemical milling (Fritsch Pulverisette7 premium line). The initial precursors, lithium sulfide , lithium bromide , and phosphorus pentasulfide, were purchased from Merck andSigma-Aldrich. All precursors were mixed in an appropriate stoichiometricratio using a mortar and pestle. The obtained 1.0 g of the precursorwas then mechanochemically milled (the milling media to precursorsratio is 30:1) using 10 mm-diameter ZrO2 balls at 510 rpmfor 25 h (every 10 min milling and 10 min rest). After each cycleof milling, the direction of rotation was reversed to achieve bettermixing of the precursors. The powder was pressed into a pellet andthen placed inside a quartz ampule. Before filling, the quartz ampuleswere heated in a Buchi at 473 K under vacuum for 12 h to remove tracesof water. The quartz ampules were sealed under vacuum (<10–3 mbar). The sealed quartz ampules were placed insidea furnace for crystallization, where a ramping rate of 100 K/h wasapplied to reach a temperature of 550 °C for x = 0.0 and 430 °C; for x = 0.3 and 0.5 (Lix6–PSx5–Brx1+). After a reaction time of2 h, two different cooling methods were applied: (1) fast coolingby quenching in liquid nitrogen or (2) slow cooling using a coolingrate of 4 K/h during 5–6 days. The final obtained powder washand-ground and stored in an argon-atmosphere glovebox. The phasepurity, lithium substructure, and ionic transport of the argyroditeseries were analyzed by X-ray diffraction, neutron powder diffraction,electrochemical impedance spectroscopy, and NMR spectroscopy.Li1 = 1.540598 Å and λ2 = 1.544426 Å,at 45 kV and 40 mA). Measurements were taken in the 2θ rangebetween 10 and 90°. All powders were placed in an airtight sampleholder with a Kapton lid under an argon atmosphere to prevent airexposure.X-ray diffraction was carried outto determine the phase purities and relevant structural parameterswith an XˈPert Pro X-ray diffractometer  in Bragg–Brentanoθ–θ geometry with Cu Kα radiation (λ45 operatingat room temperature and aneutron wavelength of λ = 1.667 Å(selected using the 533 reflections of the germanium monochromator)For each sample, approximately 1.5 g was loaded into a 6 mm-diametercylindrical vanadium can under an argon atmosphere and then sealedusing an indium wire to prevent air exposure during transport. Thesample was measured for 6–18 h from 10° < 2θ≤ 160°, under vacuum. A reference measurement of the emptysample can was subtracted from each of the reported diffractograms.Neutron powder diffractiondata were collected on a PEARL neutron powder diffractometer at theresearch reactor of the TU Delft,48 The crystallographic information file (CIF) obtained from Rietveldrefinement of the neutron diffraction data of the Lix6–PSx5–Brx1+ argyrodite series was used to visualize the lithiumsubstructure. An input file (.cube) was generated in softBV, and theBVSE landscape was plotted in VESTA50 tovisualize the Li-ion migration paths across the unit cell. More detailsof the BVSE model can be found in refs .Analyses of the impedance data were performed using RelaxIS 3 impedancespectrum analysis software.Temperature-dependentelectrochemical impedance spectroscopy was performed using an AutolabPGSTAT with an EC10 M impedance analyzer using 0.01 V amplitude andfrequency ranging from 10 MHz to 1 Hz to determine the ionic conductivityand activation energy of the samples. Approximately 200 mg of powderwas pressed into pellets with 10 mm diameter using 300 MPa of pressurefor 1 min. Stainless steel disks were attached on both sides of thepellet.50 was used to perform Rietveld refinements ofX-ray diffractionand neutron diffraction data. As a starting point for this study,the structural information obtained by the neutron powder diffractionof Li6PS5Br from ref  and Br– (Wyckoff 4a) anions since these twoanions can be exchanged. The occupancies of Br– andS2– on the Wyckoff 4a and Wyckoff4d sites were constrained to 1 (occupancies of Br– (4a) + S2– (4a) = 1, occupancies of S2– (4d) + Br– (4d) = 1), assuming fulloccupancy of these sites by S2– and Br–. The stability of the refinements was ensured by allowing the refinementof multiple correlated parameters simultaneously. Finally, lithiumoccupancies at the possible interstitial sites were determined. Alllithium positions and occupancies were fitted during Rietveld refinementwhere the starting conditions from Gautam et al.39 were used as a reference for both the lithium positionand occupancy. When a negative value for the thermal displacementparameter or occupancies occurred, the lithium occupancy was assumedto be below the detectable limit and the specific position was notincluded in the refinement model. The Fourier difference map was calculatedto identify visible indications of the Li occupation at particularsites. The overall Li+ content was constrained to maintaina charge balance with the refined Br content. The constraints on individualanion/cation occupancies are available in Tables S2–S7.TOPAS softwarefrom ref  was used6Li NMR spectrawere obtained on a Bruker Ascend 500 magnet with a static magneticfield B0 = 11.7 T equipped with a NEOconsole, corresponding to a 6Li Larmor frequency of 73.6MHz. Sample powders were packed into zirconia rotors of 4 mm diameter,and a MAS speed of 5 kHz was used. The spin–lattice relaxationtime (T1) was determined using a saturation-recovery pulse program.51 The 90° pulse and T1 were determined foreach sample individually. A recycle delay of 5 times the T1 was usedfor the single-pulse spectra to ensure full relaxation. All spectrawere referenced against LiCl in water at 0 ppm.Solid-state 6PS5Hal, where Hal– represents Cl, Br, or I, exhibits structural disorderbetween Hal– and S2– on the 4d positions, where higher disorder has been shown to resultin higher Li-ion conductivities.44 Recently,it was discovered that structural disorder can be tuned in single-compositionLi6PS5Br via the synthesis method.39 Furthermore, replacing S2– with Br– has been shown to increase the ionicconductivity. However, the effects of this substitution on the lithiumsubstructure and structural disorder are not well understood. In orderto fully understand the influence of Br substitutions in lithium argyroditesand the role of the Br–/S2– disorder,a mechanochemical method was explored to synthesize Lix6–PSx5–Brx1+ materials (x ≤ 0.5), wherethe initial precursors  were mechanically milled to ensure mixing, as reportedpreviously,38 and subsequently subjectedto heat treatment at different temperatures (550 °C for x = 0.0 or 430 °C for x = 0.3 and0.5) for 2 h, which was sufficient to achieve crystalline lithiumargyrodite. In order to control site disorder , two distinct cooling methods were applied:(1) The obtained precursors were slowly cooled at a rate of 4 °C/hover several days to minimize the Br occupancy on the 4d site in each composition. (2) The obtained precursors were quenchedin liquid nitrogen (fast cooling) to achieve a higher Br occupancyon the 4d site. The quenching was expected to “freezein” the higher Br occupancy on the 4d sitethat was achieved at higher (synthesis) temperatures.Lithiumargyrodite Lid and 4a), sites , and thermal parameters were refined. 6PS5Br; all structural tables can be found in Tables S1–S7. Samples  contained less impurity phases of Li3PO4 andLiBr (less than 1.0 wt %), but these were assumed to have negligibleimpact on the ionic transport or structural analysis. While increasingthe Br content up to x = 0.5, the cubic polymorphswere maintained. Hu et al.21 showed thatincreasing the Br content in Lix6–PSx5–Brx1+ (x = 0.7 and 0.8) led to the increase inLiBr and Li3PS4 impurities, indicating a solubilitylimit. Therefore, the analysis focused on the cubic polymorphs withinthe solubility limit (x ≤ 0.5).Rietveldrefinement of the neutron diffraction patterns was used to determinethe structure, especially with the aim to reveal the lithium substructureand the Br site disorder. The lattice parameter, occupancies Å, consistent with previous studies.39x), the lattice parameter decreasesfor the slow cooling method and increases for the fast cooling method.The difference in the systematic change in the lattice parameter asa function of the Br content and two cooling methods is noteworthyand unexpected. a site. Variation in the Br occupancyon the 4d site in Li6PS5Brcan affect the lattice parameter due to the difference in ionic radiibetween bromide (196 pm) and sulfur (184 pm)52 anions occupying the 4a sites and changes in theLi+ occupancy around the 4d/4a sites as divalent sulfur is replaced by monovalent bromine.39 For the slow cooling method, increasing theBr content does not affect the Br occupancy at the 4a site significantly . Hence,the decrease in lattice parameter with increasing Br content may bedue to the increase in Li+ vacancies or differences inLi+ distribution. In the quenched sample, a clear trendwas shown between the lattice parameter and the Br occupancy in the4a site. As the Br content increases, the Br occupancyin the 4a site also increases , resulting in a larger lattice parameter. Thistrend is consistent with the previous studies.39The Pawley-fit-refined latticeparameter of slow-cooled Lid sitecan be tuned ina single composition by adjusting the synthesis method.53 In this work, we employed a similar approach while increasing theBr content in lithium argyrodites. As the Br content (x) is increased, Br is distributed across the Wyckoff 4a and 4d sites in the crystal structure. d site obtained by the two differentcooling methods as a function of the total Br content. Slow-cooledsamples have a lower Br 4d site occupancy, as determinedby Rietveld refinements against neutron diffraction patterns. As thetotal Br content increases, Br in the 4d site increases,starting from 12% at x = 0.0 and reaching 63% at x = 0.5 for the slow-cooled method. The quenched methodhas an even higher Br in the 4d site, starting from39% at x = 0.0–72% at x =0.5, most likely because fast cooling “freezes in” thehigher Br occupancy of the 4d site that is achievedat higher temperatures.The Br occupancy on the 433 These tetrahedra areclassified based on the number of shared corners, edges, or faceswith adjacent PS43– units, and are referredto as types 1 through 5 (T1–T5).37 Li+ occupies only the type 5 and type 2  positions in Li6PS5Hal ,43 forming cage-likestructures around the central anion (Wyckoff 4d),as shown in + canpartially occupy the T4 site , the refined thermal displacement parameters ofthe T2 sites were relatively large compared to other Li+ sites in the structure , indicating a delocalizationof Li+ around the T2 site. This high value is consistentwith literature.54 The refined thermal displacement parametersof the T4 site were unphysically negative values for the slow-cooled x = 0.0 compositions. Additionally, a Fourier differencemap showed no visible evidence of Li occupation on the T4 site. Whenthe Br content increases in slow-cooled compositions up to x ≥ 0.3, additional T4 lithium sites were found tobe occupied, as shown in Figure S2. Thesesites lead to a stable and improved fit .As mentionedabove, in the lithiumargyrodite structure, the anion framework creates 6 different typesof tetrahedral interstices, five of which can potentially accommodatelithium.56 Masuda et al.40 have reported on theiroccupation in mixed-halide argyrodite systems. The occupation of theT4 site, located between two cages, has been suggested to improveLi-ion diffusion in argyrodites. There are two intercage pathwayswhich are important for long-range Li+ diffusion, as suggestedin the literature:35 one pathway throughtwo neighboring T2 sites (T2–T2) and another pathway betweentwo T5 sites via the T4 site (T5–T4–T5), as shown in Figure S4 shows that the T2, T5, and T4 polyhedral volumesare barely affected by a change in the Br content. The BVSE landscapesof the Li6–xPS5–xBr1+x series reveal that cage-to-cage Li+ diffusion pathwaysare more connected for higher Br content . 6PS5Br compositionallows only for lithium-ion diffusion through T2–T2 pathways,where no occupancy of the T4 site is observed. As the bromine contentincreases, the T2–T2 distance remains constant for the slow-cooledmethod; however, it decreases for the quenching method. The T5–T4–T5distances decrease with increasing bromide content in both synthesismethods, as depicted in x = 0.3 (composition) to 3.6 (9) Å at x = 0.5. In the quenched method, the T5–T4–T5distance decreases from 3.64 (12) Å at x = 0.0to 3.49 (8) Å at x = 0.5, as seen in The T4sites have previously been identified in Cu and Ag argyrodites.a and 4d sites will vary based on their occupancydue to the differentcharges of Br– and S2–. This canbe anticipated to change the average surrounding Li+ substructure(occupancies). To quantify how Br– impacts the arrangementof Li+, the average lithium distribution (denoted as Rmean) in the structure was determined by calculatingthe average distance between the 4d site  and Li+ located at differentsites , weighed by the occupancy of each site withina single cage, as previously introduced.39 Previously, only the T5, T2, and T5a sites were considered,39 but at present the additional T4 site is includedin the Rmean calculations. The visualrepresentation of Rmean can be seen in d Br– occupancy,significant changes of Rmean are observed were fitted with a resistorand constant phase element (CPE) in parallel; another CPE in the seriesrepresents the blocking electrode. The resistor represents the overallionic conductivity. At higher temperatures (10 and 25 °C), theresponse for the CPE/resistor shifts to higher frequencies and isdifficult to resolve within the measured frequency range. In thiscase, only the tails of the blocking electrodes were used for thefit. The CPE in parallel exhibited a capacitance of around 1 to 8× 10–10 F/cm2, indicating bulk transport.39 The impedance Z of CPE is givenby J is the imaginary unit, Q is a parameterrelated to the magnitude of the impedance, and α representsthe phase constant. The α value of 0.89–0.97 representsthe ideality of the semicircle, with an α value of 1 indicatingan ideal capacitor. d Br occupancy) leads to an improvementof the ionic conductivity.39 In this work,the conductivity of the slow-cooled Li6PS5Brsample (12% Br occupancy on the 4d site) is 0.78mS/cm. The conductivity of the slow-cooled samples increases from0.78 to 6.2 mS/cm with increasing Br content, while the conductivityof quenched samples increases from 3.21 to 8.55 mS/cm with increasingBr content. 63 This rule states that an increasein the Arrhenius prefactor can lead to an increase in the activationbarrier, as demonstrated in Figures S6 and S7, effectively meaning that more jumps occur, presumably because ofthe more optimal Li+ distribution over the sites, providinga higher conductivity.De Klerk et al.d Br occupancies through the synthesis method cansignificantly improve the ionic conductivity in lithium argyrodites.The occupancy and diffusion pathway and kinetics of lithium, withinthe Li cage and between the Li cages, are dictated by the halogencontent and distribution over the 4d and 4a sites. In this study, we were able to tune the Br occupanciesin the 4d site and the lithium substructure in eachcomposition, leading to the following observations:a site, but decreasing the Li content and differences in Li+ distribution leads to a decrease in the lattice parameter. As theBr content increases in the quenched sample, the Br occupancy at the4a site increases, which leads to a larger latticeparameter.The slow cooling method leads toa decrease in the latticeparameter, while the fast cooling method leads to an increase in thelattice parameter as the bromide content increases. The slow coolingmethod does not affect the Br occupancy at the 4d Br occupancies also increase, starting from12 to 63% for the slow-cooledmethod. The 4d Br occupancy is larger at higher temperaturesand quenching “freezes in” the higher 4d Br occupancies at room temperature.53 The quenching method therefore results in even higher 4d Br occupancies, ranging from 39 to 72%. Overall, this work highlightsthe importance of the synthesis methods as a tool to control the 4d Br occupancies in lithium argyrodites, which plays a keyfactor in the Li-ion conductivity.As the total Br content increases,the 4+ jumps through face-sharingtetrahedral environments. This alternative pathway is in additionto the T2–T2 jumps that are observed in most lithium argyrodites.The intercage distance, which is connecting neighboring lithium cagesvia the T5–T4–T5 or T2–T2 distance, appears crucialto increase the ionic conductivity. Consistently, the BVSE analysesshow that cage-to-cage Li+ diffusion pathways are moreconnected for a higher Br content .Inthe lithium substructure, the observation of nonzeroT4 occupation implies the activation of a new pathway for Li-ion diffusion,consisting of shorter intercage Lid Br occupancyincreasesfor both cooling methods (see above section), the average negativecharge on the 4d site decreases as the divalent sulfideis replaced by the monovalent bromide ion. This reduces the electrostaticinteraction between the electronegative 4d site andthe electropositive Li ions. The Li ions move away, causing Rmean to increase and subsequently, more chargeis maintained on the Li ion, causing the observed 6Li shiftto move to lower ppm values. The increased Li cage radius , resulting in anincrease in ionic conductivity from 3.21 to 8.55 mS/cm. Hereby, wehighlight how the bromide content and sublattice occupancy affectthe Li cage expansion and the Li occupancies, which can be used toimprove the diffusion pathway and Li-ion mobility and thus the Li-ionconductivity. We anticipate that understanding these structure–conductivityrelationships further enhances the application potential of lithiumargyrodites in solid-state lithium-ion batteries.Increasing the4x6–PSx5–Brx1+) with compositions rangingfrom x = 0.0 to 0.5 using two cooling methods. Itis found that the cooling method controls the Br occupancy on the4d site in the Lix6–PSx5–Brx1+ series. Rietveld refinement of the neutron diffraction patternsreveals how this affects the 4d and 4a site occupancies and the lithium substructure. As the Br contentincreased, the Li+ ions moved away from the center of thecage (4d site), as captured by the Rmean. Additionally, this induces T4 site occupancy, whichopens up an alternative Li-ion diffusion pathway with a lower activationenergy. The average negative charge on the 4d sitedecreases due to an increase in the 4d Br– occupancy,resulting in an increased Li cage radius and reduced T2–T2and T5–T4–T5 distances, leading to a better connectedLi+ landscape. The slow-cooled method results in an increasein ionic conductivity from 0.78 mS/cm at x = 0.0–6.2mS/cm at x = 0.5, while the quenched method showsan increase from 3.21 mS/cm at x = 0.0–8.55mS/cm at x = 0.5. These findings demonstrate theimportance of understanding how chemical changes and synthesis conditionsaffect the lithium substructure in order to tailor the transport propertiesof these materials and increase their potential for solid-state batteries.In this study, we synthesized bromide-enrichedlithium argyrodites(Li


Jimusaria is the first reported Chinese dicynodont, previously only known from Xinjiang. Here we refer two specimens from the Naobaogou Formation, Nei Mongol, China to Jimusaria based on the following features: squamosal separated from supraoccipital by tabular, tabular contacting opisthotic, sharp and thin lateral dentary shelf expanding anteriorly into a thick swelling, nasals fused as single element, rod-like medial bar formed by footplate of epipterygoid connecting to the parabasisphenoid and periotic medially. A new species, J. monanensis, is named based on the diagnostic characters on these two specimens such as distinct caniniform buttress lacking posteroventral furrow, naso-frontal suture forming an anterior directed sharp angle, and converging ventral ridges on posterior portion of anterior pterygoid rami. In Jimusaria, the epipterygoid posteromedially contacts the parabasisphenoid and the periotic as a rod-like bar, a unique morphology unknown in any other dicynodonts. This structure probably increases the stability of the palatal complex. A similar structure might also appear in other dicynodonts as a cartilage connection. The new occurrence of Jimusaria increases the diversity of the tetrapod assemblage from the Naobaogou Formation, and further strengthens the connection between the tetrapod faunas from Nei Mongol and Xinjiang. Based on the current record, Jimusaria is one of the few tetrapod genera which survived in the end-Permian mass extinction. Dicynodon sinkianensis is the first named species from those specimens, and is the first record of Chinese Permian-Triassic tetrapods. Later, this species was transferred to a new genus, Jimusaria, resulting in a new combination, J. sinkianensis ; the skull basal length is 223 mm  and 2. IBiarmosuchus served as the outgroup to root the most parsimonious tree in our analyses. Symmetric resampling values were calculated based on 10,000 replicates.The matrix is based on the recent works . The finhttp://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:B5D7A3C5-1C07-4F71-BC24-9141CD5B658E. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central SCIE and CLOCKSS.The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix Anomodontia Owen, 1860Dicynodontia Owen, 1859Dicynodontoidea Olson, 1944JimusariaSun, 1963Genus Type species.Jimusaria sinkianensis  (oracoid)  and 2.Type locality and horizon. Tumed Right Banner, Nei Mongol, China; Member I, Naobaogou Formation.Referred material. IVPP V31929 Shiguai, Baotou, Nei Mongol, China. Member I, Naobaogou Formation, an incomplete skull with complete mandible J. sinkiaThe parabasisphenoid bears paired internal carotid canals, whose ventral openings are located near the suture with the pterygoid . The parKingoria  as the sister group of Kannemeyeriiformes  a cartilaginous connection is present between the quadrate ramus of the pterygoid and the lateral wall of braincase in most if not all dicynodonts, but the ossification occurred in Jimusaria. The second scenario seems more plausible considering the inferred function of the epipterygoid. The cartilaginous connection can provide a weaker but similar support function.The epipterygoid ossified from the palatoquadrate cartilage; representing an element of the splanchnocranium, in early tetrapods it connects to the quadrate frequently . In dicyJimusaria sinkianensis  has a smoother snout region with only some pits and rugosity , while tianensis –4D. The gdaensis .Jimusaria monanensis.Comparing two specimens (IVPP V26034 and V31929), the interorbital region becomes relatively wider, and the intertemporal bar becomes narrower ; the pterygoid medial plate is relatively longer while the interpterpterygoid vacuity is relatively smaller in the bigger specimen (IVPP V26034). The smaller specimen (IVPP V31929) already has a distinct caniniform buttress, indicating that feature developed at a relatively young age in J. sinkianensis, three small specimens (IVPP V3240) show the distinct caniniform buttresses and the indistinct posteroventral furrow continues dorsoposteriorly to the depressions in the maxilla; the larger specimen (IVPP RV341407) possesses fatter caniniform processes with distinct posteroventral furrows and stronger depressions (In ressions . The tusTurfanodon jiufengensis implies the Naobaogou Formation shares common taxon with the Guodikeng Formation (Jimusaria sinkianensis, T. bogdaensis, Diictodon feliceps, and some Lystrosaurus species (J. monanensis enhances the connection between the faunas of Nei Mongol and Xinjiang. Same as Turfanodon, Jimusaria is the second reported dicynodont genus which has a distribution across the temperate and tropic zones.The tetrapod assemblage of the Naobaogou Formation is the most diverse tetrapod assemblage of the Lopingian in China, the previous report of ormation . Several species . The finJ. sinkianensis is the upper portion of the Guodikeng Formation, early Triassic in age (J. monanensis were collected from Member I of the Naobaogou Formation, which possibly correlates with the Wuchiapingian (Jimusaria is a rare example of a tetrapod genus which survived in the end-Permian mass extinction.Recent stratigraphic work placed the Permo-Triassic boundary at the base of the Guodikeng Formation in Xinjiang . Based oc in age  while otapingian , 2021. SJimusaria monanensis, was discovered in the Naobaogou Formation, this record further increases the diversity of the tetrapod assemblage of this formation and the connection between the North China and Xinjiang during the Lopingian, Permian. The peculiar rod-like medial bar of the epipterygoid in Jimusaria likely performed a function in stabilizing the palatal complex, and a similar structure may have been present in many other dicynodonts as part of palatoquadrate cartilage remnants. Jimusaria spanned from the Wuchiapingian to the Early Triassic in age.A new dicynodont species, 10.7717/peerj.15783/supp-1Supplemental Information 1Click here for additional data file.


Wave and sea ice properties in the Arctic and Southern Oceans are linked by feedback mechanisms, therefore the understanding of wave propagation in these regions is essential to model this key component of the Earth climate system. The most striking effect of sea ice is the attenuation of waves at a rate proportional to their frequency. The nonlinear Schrödinger equation (NLS), a fundamental model for ocean waves, describes the full growth-decay cycles of unstable modes, also known as modulational instability (MI). Here, a dissipative NLS (d-NLS) with characteristic sea ice attenuation is used to model the evolution of unstable waves. The MI in sea ice is preserved, however, in its phase-shifted form. The frequency-dependent dissipation breaks the symmetry between the dominant left and right sideband. We anticipate that this work may motivate analogous studies and experiments in wave systems subject to frequency-dependent energy attenuation. The sea ice properties are intimately linked to ocean wave properties via feedback mechanisms in the marginal ice zone (MIZ)7 which around Antarctica, fed by intense Southern Ocean waves all year round8, extends for hundreds of kilometers11. Rapid evolution of the polar regions driven by climate change14 have revived and energised research activities in understanding waves properties and feedback in the MIZ15, including in the emerging Arctic MIZ16.Arctic and Antarctic sea ice play a prominent role in the Earth system by regulating heat and momentum exchanges over large spatial scales18, as shown in Fig. 21. In the MIZ interior, where floes are larger and comparable to the wavelength, wave attenuation by scattering dominates20. At leading order in wave steepness, i.e. the wave nonlinearity parameter, each wave component attenuates exponentially with distance, see schematic in Fig. 23. That is, shorter waves are attenuated faster than their longer counterparts. For a comprehensive review of waves in sea ice we refer the reader to Meylan et al.19 and Squire20, and references therein.In the MIZ exterior, where the sea ice cover is a mixture of small floes (much shorter than the wavelength) and interstitial frazil ice24. In fact , and in contrast to the linear stability analysis of Stoke waves, the complete growth and decay cycles can be described within the NLS framework25. More recently, several studies have been devoted to investigate the effect of wave dissipation on the phase-shifted recurrent MI focusing cycles31. The latter studies highlight the phase-shifted recurrence in the long-term evolution of nonlinear and unstable waves, when constant, weak, and linear dissipation effects are at play. That said, the NLS can be also adapted to accommodate the influence of sea ice attenuation on the waves by including viscous-like losses as a dissipative term that matches the decay rate of the linear amplitude, as shown by33. Within this context, it as been shown that it is important to account for the ice-induced frequency dependency in the attenuation of ocean waves in the MIZ34.Narrowband ocean wave dynamics can be accurately described by the nonlinear Schrödinger equation (NLS). One intriguing dynamical phenomenon, which is responsible for the formation of large-amplitude and coherent waves and has attracted the scientific interest since the late 60s, is the modulation instability (MI)34]:x, while t denotes the time in the frame of reference moving with at the group speed , and usually falls in the range 2–419. The same applies for the real coefficients 19 and, therefore, on effective sea ice properties 40. The power law dissipation with exponent 19, and will be therefore considered herein. The exponent n is derived from the classical linearised water wave problem when an extra pressure term is added to the free surface dynamic boundary condition to model sea ice. When the pressure term is assumed proportional to the vertical velocity the resulting wave dispersion relation has imaginary part  proportional to 19. Note that the model is equivalent to the one discussed in34, but in this case the dissipative coefficients Following41.We investigate the classical MI problem of a monochromatic wave train with frequency 42]:18, wave conditions few tens of kilometers away from the sea ice edge and within the MIZ are illustrated in Fig. The wave steepness, defined using wavenumber of the carrier wave and the sum of all amplitudes, . However, it is worth noting that for a continuous spectrum the carrier wave component progressively shifted to longer wave period50 which are also less dissipative. On the other hand, the three wave system has a more symmetric spectrum and the period of the carrier wave component remains constant during the long-term evolution in space. It is also noteworthy that for the lowest dissipation level, at the end of the spatial domain in our numerical simulations (approximately three recurrence cycles), the mean wave amplitude is reduced by less than 1%. Nonetheless, the spatial pattern of the recurrence cycles still becomes phase-shifted, see Fig. The spatial evolution of the mean wave amplitude mentclass2pt{minimIn each of the simulations performed, we tracked the maximum amplification in space, irrespective of time, i.e. entclass1pt{minimaThe evolution of the carrier wave  to A–Type (shifted) breather-type evolution. The most important effect of the differential attenuation rate modifies the dynamics of the left/right sidebands and there is a lag between the two. However, compared to frequency-independent attenuation, differences in energy and recurrence pattern appear unaffected. The large initial nonlinearity of the system triggers pseudo-recurrence cycles with large amplification of the initial sea state, particularly, for low and medium dissipation. Interestingly, for medium dissipation rates the shortening of the cycle means that there are more instances in the domain with amplifications greater than two, compared to the conservative case. For comparison, random sea states, see Ref.34, reverted towards Gaussian statistics in presence of dissipation.The classical dynamics of the modulational instability is studied in the presence of a frequency-dependent attenuation, this is in contrast with previous works in which forcing and damping were considered frequency independent, e.g.34) in which for large dissipation make a considerable difference between the linear and nonlinear case. This difference is attributed to the shift of the peak frequency in a continuous wave spectrum, whereas for the classical MI case the carrier wave frequency remains unshifted.Despite the complex dynamics in the nonlinear evolution, the total energy decays in agreement with linear theory, this contrasts the random waves  to very high attenuation values. Recalling that mentclass2pt{minimIt is worth noting that dissipation scales The d-NLS  is solve


Minimally invasive spine surgery is a field of active and intense research. Image-guided percutaneous pedicle screw (PPS) placement is a valid alternative to the standard free-hand technique, thanks to technological advancements that provide potential improvement in accuracy and safety. Herein, we describe the clinical results of a surgical technique exploiting integration of neuronavigation and intraoperative neurophysiological monitoring (IONM) for minimally invasive PPS.An intraoperative-computed tomography (CT)-based neuronavigation system was combined with IONM in a three-step technique for PPS. Clinical and radiological data were collected to evaluate the safety and efficacy of the procedure. The accuracy of PPS placement was classified according to the Gertzbein–Robbins scale.A total of 230 screws were placed in 49 patients. Only two screws were misplaced (0.8%); nevertheless, no clinical sign of radiculopathy was experienced by these patients. The majority of the screws  were classified as grade A according to Gertzbein–Robbins scale, seven screws were classified as grade B, one screw was classified as grade D, and one last screw was classified as grade E.The proposed three-step, navigated, percutaneous procedure offers a safe and accurate alternative to traditional techniques for lumbar and sacral pedicle screw placement.Level of Evidence Level 3.Trial registration Not applicable. Minimal invasive spine surgery has been shown to result in lower rates of complications such as infection and bleeding, reduced postoperative pain, shorter hospitalization, and decreased mortality compared with open surgery , 19. PerThe traditional free-hand technique needs extensive experience and skill from the surgeon; furthermore, intraoperative fluoroscopy control of the screw placement exposes surgeons and other personnel in the operating room to ionizing radiations. On the other hand, image guidance offers an augmented visualization of the anatomical landmarks without direct exposure. However, there are two major drawbacks: first, the increased radiation exposure for the patient; secondly, the risk of screw malpositioning in case of movement of the reference array. Nevertheless, image-guided screw placement is becoming increasingly popular among spinal surgeons due to the decrease in breach rate and improvements in accuracy . A recenIntraoperative neurophysiological monitoring (IONM) is increasingly used in fusion surgery. In particular, triggered electromyographic monitoring is highly specific but weakly sensitive for predicting new postoperative deficit due to pedicle breach during screw placement . In the Following approval by our local ethical committee (no. 276/120/CE), we conducted a retrospective review of 49 consecutive patients who underwent lumbar arthrodesis with PPS for degenerative conditions of the lumbosacral spine. Surgical procedures were performed by the senior author (G.S.) and his assistant (G.L.R.) and were performed at the same institution. All patients signed a written informed consent before surgery. Inclusion criteria for the study were: (1) age at surgery between 18 and 80 years old, (2) low back pain (LBP) with radicular irradiation in the lower limbs, (3) neurogenic claudication, and (4) failed conservative treatment for at least 6 months. Patients with a previous history of instrumented spine surgery were excluded from the study. Demographic, intraoperative, clinical outcome, and radiological data were recorded.step 1, Fig. step 2, Fig. step 3, Fig. All procedures were performed on a TruSystem 7000 table (TRUMPF Medizin Systeme GmbH) with a BrainLab Curve 1.2 navigation system  linked to AIRO Mobile intraoperative CT scan . Instrumentation systems were manufactured by NuVasive  except for the navigated drill guide (see ) and theAge, sex, body mass index (BMI), and length of hospital admission was recorded for all patients. Surgical time between skin incision and first CT scan (which corresponds to the time necessary to place the clamp on the spinous process), and surgical time between first and second intraoperative CT scans (which corresponds to the effective time taken to place pedicle screws) were recorded. Dose exposure was routinely recorded for each patient.The accuracy of screw placement was evaluated blindly by a senior neuroradiologist on a postoperative CT scan performed the day after the procedure, according to the Gertzbein–Robbins scale . The radStatistical analysis was performed using GraphPad Prism 5.01 software . As appropriate, radiological and clinical data were quantitatively expressed as a range, with relative medians or percentages.Forty-nine patients (22 men and 27 women) were treated between January and April 2021 at the same institution during the first 3 months of using the present navigation-guided technique, for a total of 230 screws  (Table The average absorbed radiation dose (dose length product) per patient was 1586.4 mGy/cm (range 722.8–2267 mGy/cm). The median time lapse between the two intraoperative CT scans was 29 min (range 59–16 min), corresponding to a total of 6 min and 15 s range (4 min - 14 min and 45 seconds) per screw.n = 7 (3%)], D [n = 1 (0.4%)], and E [n = 1 (0.4%)]  screws in our series were misplaced (one screw was classified as grade D and one as grade E) Table . The pos)] Table .Table 2PThe principal aim of this technical note was to report the efficacy and safety achieved with this newly described three-step technique integrating a navigated drill guide and IONM for PPS. In the present study, a perfect route (grade A) was achieved for 221 screws (96.1%) whereas 7 screws 3%) resulted in grade B. Only a minor portion of the screws (0.8%) were graded as D or E, although this did not cause any clinical issue. The accuracy of the described technique is in accordance with literature data relative to image-guided screw placement % resulte, 14, 21.Both of our misplaced screws were laterally displaced, with an “in–out–in” trajectory; the mechanical properties of the screws seemed acceptable (as far as it is possible to assess in the operating room). Moreover, the normal IONM response was an adjunctive element that led us to the decision not to replace the screw in both cases. In all cases, including those in which the screws were misplaced, the clinical outcome was uneventful. This is in line with the experience of spinal surgeons and previous literature that not all radiologically misplaced screws lead to clinically relevant consequences , 10, 23.Notably, a long experience with the free-hand technique is very useful to have as a “plan B” technique in case of malfunction of the workstation, and to recognize any eventual mismatch between intraoperative guidance and effective position of the surgical instrument. The tactile feedback of the consistency of the bone is, in fact, an immediate, real-time confirmation that reduces the need of time wasting, repeated checks of accuracy on visible landmarks. In our opinion, the use of intraoperative image-guidance does not replace the need of accurate knowledge of local anatomy and this has a fundamental importance when you decide to try a new strategy or tool. The concomitant use of IONM is an adjunctive source of safety, even though we are aware that there is no definitive evidence that it may prevent neurological deficit .The use of a midline incision for placement of the clamp on the spinous process has not implied an adjunctive incision, as all the patients in our case series underwent lamino-arthrectomy after screw placement. An important issue in this procedure is the exposure to radiation. The patient is exposed to a considerable amount of radiation, even if navigated techniques sometimes showed a lower level of exposure than 2D-fluoroscopy guided procedures . Our levSeveral new techniques have been developed in the last few years to improve precision and safety in pedicle screw insertion, such as intraoperative imaging, image-guidance with navigation, robot assistance, and IONM. Though probably not essential, these techniques can help the spinal surgeons, even the most experienced, in reducing risks for the patients. Moreover, they make the training of younger surgeons faster and safer, even if the supervision of an expert in the field is nevertheless fundamental , 10, 14.A recent meta-analysis compared the revision rate of PPS in free-hand, navigated, and robot-guided thoracolumbar instrumentation, and showed that assisted techniques (robotic or navigated) may reduce the incidence of costly and clinically relevant postoperative revisions for screw malposition .1. It is of paramount importance to secure the spinous process clamp before the CT scan. The clamp must be tightly fixed on the bone, not to the ligament. At the same time, it is necessary not to use too much force to avoid fractures of the spinous process or breaking the clamp. The soft tissue retractor is removed before the CT scan to reduce artifacts. The reference attached to the clamp must not be in contact with the skin and should have the correct inclination to be visible both at the moment of the CT scan and during screw placement. Moreover, it must not hamper the screw placement. The use of the drill guide instead of a navigated trocar has the advantage of reducing the vibrations that are transmitted to the clamp, with the risk of unintentionally moving the reference.2. IOM helps to avoid neurologically significant misplacement of the screw and gives a continuous, real-time feedback during pedicle drilling and during screw placement. An intraoperative CT scan performed after screw placement provides an immediate control of the position of the screws relative to the anatomy of the patient. The combination of neurophysiological and radiological data improves the safety of the procedure.3. This technique allows the placement of only one screw at a time, but the collaboration between the two operating surgeons and the standardization of the workflow greatly reduces the time and the risk of inaccuracy.4. Cooperation between surgeons is essential during placement of the Kirschner wire and for removal of the drill guide. This is a crucial step, usually done with four hands because it is the only “blind” step of the procedure. The risk of skiving on the bony surface, especially in case of facet bony spurs, is lowered by the presence of sharp prominences on the extremity of the drill guide, but the collaboration between surgeons remains the most important element in avoiding mistakes. The wire is inserted only 2.5 cm deep in the pedicle, as set with the drill guide, so the real risk of penetration of the wire in the abdominal viscera and major blood vessels is very low.5. Finally, usage of a K-wire has, in our opinion, two advantages: (a) the wire can be used as a feeler to confirm the bony consistence of the tissue before screw placement and (b) in case of inaccuracy of image guidance, the immediate placement of the screw in an incorrect position has a higher risk of irreversible damage to neural structures or to pedicle.Our experience with the described technique highlighted some important points to improve the safety and accuracy of the procedure:Our study/technique has some limitations. The present study was conducted on a small number of patients and only on degenerative lumbo–sacral pathology. There is a risk of skiving on the drill guide during K-wire insertion . The risIn conclusion, this study suggests that our three-step technique for PPS can be a valid option to use in minimally invasive spine surgery (MISS) procedures. It exploits advantages of neuromonitoring and CT-guided navigation. We have used this technique for implantation of more than 200 pedicle screws with no complications so far, and excellent results in terms of pedicle screws placement accuracy.


Uria lomvia) in response to broad‐scale environmental conditions at two different‐sized, low Arctic colonies located <300 km apart. First, we compared foraging behavior (measured via GPS units), average daily energy expenditure (estimated from GPS derived activity budgets), and foraging success  of murres at two colonies, which differ greatly in size: 30,000 pairs breed on Coats Island, Nunavut, and 400,000 pairs breed on Digges Island, Nunavut. Second, we tested whether colony size within the same marine ecosystem altered foraging behavior in response to broad‐scale environmental variability. Third, we tested whether environmentally induced foraging flexibility influenced the foraging success of murres. Murres at the larger colony foraged farther and longer but made fewer trips, resulting in a lower nutritional state and lower foraging success compared to birds at the smaller colony. Foraging behavior and foraging success varied in response to environmental variation, with murres at both colonies making longer, more distant foraging trips in high ice regimes during incubation, suggesting flexibility in responding to environmental variability. However, only birds at the larger colony showed this same flexibility during chick rearing. Foraging success at both colonies was higher during high ice regimes, suggesting greater prey availability. Overall, murres from the larger colony exhibited lower foraging success, and their foraging behavior showed stronger responses to changes in broad‐scale conditions such as sea ice regime. Taken together, this suggests that larger Arctic seabird colonies have higher behavioral and demographic sensitivity to environmental change.The degree to which individuals adjust foraging behavior in response to environmental variability can impact foraging success, leading to downstream impacts on fitness and population dynamics. We examined the foraging flexibility, average daily energy expenditure, and foraging success of an ice‐associated Arctic seabird, the thick‐billed murre ( Recent climate‐induced changes in sea‐ice dynamics are impacting Arctic marine ecosystems, further exacerbating the effects of colony size on foraging behavior. To assess the adaptive capacity of Arctic seabirds to cope with changing sea ice dynamics, our study investigates the foraging behavior and resulting foraging success of thick‐billed murres under different sea ice regimes at two different‐sized colonies. We found that thick‐billed murres at both colonies had lower foraging success under low ice regimes and that thick‐billed murres at a larger colony had overall lower foraging success suggesting larger colonies in the Canadian Arctic may be more susceptible to environmental change. As prey depletion rates are expected to be directly related to colony size Ashmole, , we expe22.1We conducted fieldwork at two murre colonies located within the Hudson Strait‐Northern Hudson Bay Narrows region of the Eastern Canadian Arctic: Coats Island, NU , and Digges Sound, NU . Although the sites are only 220 km apart, they vary greatly in colony size. Coats Island hosts 30,000 breeding pairs and 400,000 pairs breed on Digges Island  obtained through remote sensing . Murres are associated with sea ice , a McGill Animal Use Protocol (2015‐7599), and Environment and Climate Change Canada Animal Care and Collection permits . We captured adult murres during incubation and chick rearing using a noose pole at nest sites. We blood sampled murres within 3 minutes of capture (to ensure the measurement of baseline physiology—see below), collecting 1–2 mL of whole blood from the brachial or jugular vein using either a 26‐guage needle, heparinized capillary tube, and heparinized Eppendorf tube (Digges Island) or a 25‐guage needle, 3 mL syringe, and heparinized vacutainer (Coats Island). We kept blood samples on ice, for a maximum 8 h, and then centrifuged for 5–10 min at 2000 t et al. . We usedt et al. . If a muAfter blood sampling, we banded murres for individual identification and measured body mass (g). We attached GPS devices  to murres' dorsal feathers , at the colony , and swimming  over the deployment period to estimate average daily energy expenditure  using the equation:We processed the GPS data and extracted foraging metrics in t et al.   to collapse down the multiple foraging variables we extracted from GPS units during both the incubation and chick‐rearing stages. The incubation PCA generated a single eigenvalue greater than one, explaining 65.3% of the variation, with maximum distance, average daily distance, mean trip distance, and mean trip duration strongly positively loaded onto factor one and number of trips per day strongly negatively loaded onto factor one  at the two colonies, we used kernel density analysis. We calculated the 95% and 50% utilization distributions from foraging locations  using the Calenge, .2.5We quantified nutritional biomarkers—plasma triglycerides (TRIG), baseline corticosterone (bCORT), beta‐hydroxybutyrate (B‐OH), and non‐esterified fatty acids (NEFA) in plasma samples to obtain pre‐foraging levels, post‐foraging levels, and relative change of nutritional biomarkers (∆ = log(post‐foraging levels) – log(pre‐foraging levels)) as estimates of nutritional state and foraging success Table . To calc2.62.6.1We used a Kruskall–Wallis test followed by a pairwise Wilcox‐test with a Bonferroni p‐value adjustment on daily mean sea ice concentration and sea surface temperature for each colony, to categorize years as high ice regime years  or low ice regime years .2.6.2lme4  and the start date of the deployment were fitted as random effects to account for repeated samples of individuals (when sample size was sufficient) and to account for temporal autocorrelation, respectively (see Supplementary File). When a low number of repeated individuals precluded the use of LMMs, we fit linear models, and repeated individuals were removed to meet test assumptions. For all models, sex and year (if there were more than two years) were fitted as fixed effects to account for sex differences and interannual variation, and GPS deployment duration was also included as a fixed effect, when significant, to account for variation in deployment lengths. For nutritional state and foraging success models, time at the colony before the bird was sampled after returning from a foraging trip (TimebfSampling) was included as a fixed effect, if significant, to account for changes in physiological parameters over time.p > .05) they were removed from the model. We then re‐fit models using restricted maximum likelihood estimation (REML) and used lmertest  and conditional R2  for all models via MuMIn  to test for significance of interactions between fixed effects, if interactions were nonsignificant  and Digges Island colonies . For sea ice concentration at Coats Island, 2018 differed from both 2017 (p‐adjusted = .01) and 2019 (p‐adjusted = .01), whereas 2017 and 2019 did not differ (p‐adjusted = 1.0). For sea surface temperature at Coats Island, all years differed from each other . We therefore categorized 2019 and 2017 as warm, low ice years . For sea surface temperature at Digges Island, 2015 differed from both 2014 (p‐adjusted <.001) and 2016 (p‐adjusted <.001), whereas 2014 and 2016 did not differ (p‐adjusted = 0.78). We therefore categorized 2014 and 2016 as warm, low ice years  still varied between colonies during chick rearing, with murres at the larger colony making fewer trips and foraging farther from the colony, average daily distance traveled and average daily energy expenditure of murres did not vary between colonies. This suggests that birds at both colonies respond to the increased pressures of chick demand (while still fueling somatic needs) with different foraging strategies that nonetheless optimize mean distance traveled per day. However, the lower foraging success seen in murres at the larger colony, Digges Island, likely reflects higher interspecific competition for prey during chick rearing and negative carry‐over effects from lower payoffs during the incubation stage (lower prey availability surrounding the colony as a function of density dependence)  during high ice regimes, initially suggesting lower prey availability. However, this could reflect high flight costs associated with more distant foraging trips , has increased in the diet of black‐legged kittiwakes (Rissa tridactyla) breeding in Svalbard, Norway, with Atlantic fishes being more abundant in years with a lower sea ice index  during chick rearing was only impacted by GPS deployment duration during low ice regimes, this also suggests that there was lower prey availability in low ice regimes. Interestingly, during chick rearing, when sea ice concentration was below 10% within the local foraging range of murres at the larger colony, murres had a higher nutritional state (lower post‐foraging baseline corticosterone) in low ice regimes when average daily distance traveled was low. The opposite was observed under high ice regimes, when murres had a higher nutritional state (lower post‐foraging baseline corticosterone) when average daily distance traveled was high. This suggests murres could be foraging on different prey items under different ice regimes, where one strategy is successful during high ice regimes, and a different strategy is successful under low ice regimes. As previously mentioned, under high ice regimes (when ice is still present during the breeding season), murres may be foraging on distant sympagic arctic cod, and murres exhibiting this foraging strategy were most successful, as baseline corticosterone was lowest when average daily distance was highest. Under low ice regimes, baseline corticosterone was lowest when murres had lower average daily distances traveled, suggesting murres that foraged for coastal prey were more successful. Notably, the proportion of sub‐arctic Atlantic fishes, e.g., capelin and Atlantic herring  has been shown to impede foraging of murres breeding at high Arctic sites, e.g., Prince Leopold Island  in response to sea ice regime during the chick‐rearing stage, likely reflecting the high cost of chick provisioning, constraining foraging closer to the colony. Regardless of environmental conditions, lower intraspecific competition surrounding a small colony may leave adequate prey resources during the chick‐rearing stage to allow for closer foraging. Furthermore, as adults must maximize foraging for chicks, they may switch to more benthic or invertebrate prey that is found at closer distances ; data curation ; formal analysis (lead); funding acquisition (supporting); investigation ; methodology ; visualization (lead); writing – original draft (lead); writing – review and editing . Allison Patterson: Data curation ; formal analysis (supporting); methodology (supporting); writing – review and editing . Graham Sorenson: Conceptualization ; data curation ; funding acquisition (supporting); investigation ; writing – review and editing . Thomas Lazarus: Data curation ; writing – review and editing . Shannon Whelan: Data curation ; formal analysis (supporting); writing – review and editing . Oliver P. Love: Conceptualization ; data curation ; formal analysis (supporting); funding acquisition ; investigation ; methodology ; project administration ; resources ; supervision (lead); writing – review and editing . Kyle H. Elliott: Conceptualization ; data curation ; funding acquisition ; project administration ; resources ; writing – review and editing . H. Grant Gilchrist: Conceptualization ; data curation ; funding acquisition (lead); investigation ; project administration ; resources ; writing – review and editing .GPS tracking data will be made available on Movebank, a public repository of tracking data.Data S1Click here for additional data file.Table S1‐S12Click here for additional data file.Table S13‐S18Click here for additional data file.Table S19‐S23Click here for additional data file.


Using antibacterial agents to remove the foul odor of the implant cavity and prevent peri-implantitis can affect the detorque values and lead to the loosening of the abutment screw. This study investigated the effects of tetracycline and chlorhexidine gel on detorque values.2 and then loosened. Finally, the detorque values were calculated.This in vitro study was carried out on three groups of five implants. Group G1 was the control group, and no material was applied to the implant cavity. In group G2, the implant cavity was first filled with artificial saliva and then with chlorhexidine gel. In group G3, the implant cavity was first filled with artificial saliva and then with tetracycline. The abutments were tightened with 25 N/cmP<0.05) between the three groups.The highest detorque values were recorded in group G1. Group G3 showed the lowest detorque values. ANOVA showed significant differences in mean detorque values . According to recent studies, the success rate of implant treatments is 97‒99%, which has led to the increasing use of dental implants. However, mechanical and biological problems that lead to implant failure continue to be reported. Mechanical complications include screw fractures, implant fractures, and restoration fractures. Biological complications include excessive force on the bone, bone resorption, and microflora diffusion in microgaps between the implant and abutment.Dental implants have become one of the most widely used treatment modalities in modern dentistry, and advances in implantology have significantly improved the quality of treatments offered to patients. The incidence of screw loosening varies depending on the type of restoration, and the highest rate was observed with single crowns. In other studies, the screw loosening rate was around 12.7% for single crowns and 6.7% for fixed partial dentures. Inadequate preload, screw shape, cantilever structures, occlusion scheme or incorrect crown anatomy, bone remodeling, and bruxism are some of the reasons for screw loosening.Among the mechanical complications, screw loosening is the most important regularly reported complication. It can cause micromotion and other problems, such as inflammation of the soft tissue around the implant (peri-implantitis) and breakage of the screw. Implant systems with screw retention modalities have been used with high success rates for years. In this construction, supra structures are connected to the implant body with a titanium screw. The presence of inflammation at the implant‒abutment level affects the implant’s durability and jeopardizes its survival. Microleakage at the implant‒abutment connection is the most important cause of the inflammatory reaction around the implant. Microleakage leads to bacterial colonization around the implant‒abutment complex, which leads to the onset of the pathophysiological process of bone loss followed by implant loss.The colonization of the microflora and the presence of microorganisms in the microgap of the implant and abutment cause an unpleasant odor for the patient and the restorative dentist when the healing abutment is opened during prosthetic appointments. In addition, bacterial infection can lead to bone loss and disrupt the osseointegration process in the postoperative repair phase. The penetration of microorganisms can occur during the opening and closing of the abutment or after loading the implant.The penetration of microorganisms into the microgap can lead to inflammation in the soft tissue around the implant (peri-implantitis) and mucositis around the implant (peri-implant mucositis). using an antimicrobial agent such as chlorhexidine on the implant cavity before covering the screw or healing abutment may reduce the accumulation of bacteria and the leakage of bacteria and toxins into the implant cavity. Another study showed that applying 1% chlorhexidine gel to the implant cavity before the abutment was placed over six months led to a significant reduction in bacterial colonization in the implant cavity. The presence of these antibacterial agents can cause slippery surfaces, ultimately affecting the torque and post-rotation of the abutment screw. In one study, applying 0.2% chlorhexidine gel to the implant cavity reduced the amount of detorque and preload and increased the risk of screw loosening.To prevent these possible side effects, the use of antibacterial agents is recommended. According to a study by Micarelli et al, According to other studies, the presence of saliva has no significant effect on the level of torque and detorque. Another study found that placing zirconia abutments in the implant cavity in the presence of saliva had a greater degree of detorque than placing abutments in the dry implant cavity. However, the effect of other oil- and water-based antibacterial agents on torque and detorque values has not been evaluated.Nevertheless, the drying out of the implant cavity can reduce the tension when tightening the abutment screw, increasing the likelihood of screw loosening, loosening of the prosthesis, screw breakage, and peri-implantitis. Considering the limited studies on the effects of various antibacterial agents on the torque and detorque values of abutment screws, different results have been reported on changes in the detorque values, and there are contradictions regarding the change in the detorque values. Therefore, this study measured and compared the detorque values of abutment screws with two types of oil- and water-based antibacterial agents. the means ± SD of the abutment screw detorque in the control and chlorhexidine groups were considered 19 ± 0.43 and 17.28 ± 0.39, respectively, with α = 0.05 and β = 0.80. Therefore, the sample size was calculated at four implants in each group. However, to increase the study’s validity, five implants were included in each group, totaling 15 implants.According to a study by Jo,Fifteen DIO UF cylindrical fixtures  with a diameter of 41 mm and a length of 11.5 mm were divided into three groups of five . In addi2 and returned to the container. After one week, the implants were retrieved from the saliva container, the healing abutments were removed, and the abutments were replaced and tightened with a torque of 25 N/cm2.In group G1, the implant cavity was only soaked with artificial saliva . After one week, the implants of this group were retrieved from the container of artificial saliva, their cover screw was removed, and the implant cavity was filled with artificial saliva. Then the healing abutment was tightened to 15 N/cm2 and returned to the container. After one week, the implants were retrieved from the saliva container, the healing abutment was removed, and the abutment was reinserted and tightened with a torque of 25 N/cm2.In group G2, the implant cavity was first soaked with artificial saliva and then with 0.2% chlorhexidine . After one week, the implants in this group were retrieved from the container, their cover screw was removed, and the implant cavity was filled with 0.2% chlorhexidine. Then the healing abutment was tightened to 15 N/cm2 and returned to the saliva container. After one week, the implants were retrieved from the saliva container, the healing abutment was removed, and the abutment was reinserted and tightened with a torque of 25 N/cm2. Due to the influence of the screw tightening speed on the torque value, the screws were closed with a digital torque meter . The load applied by the digital torque meter was controlled, and the exact torque required to tighten the screw was displayed on the device monitor . The implant cavity was filled with 3% tetracycline, and then the healing abutment was tightened with 15 N/cm monitor . Due to P < 0.05 was considered statistically significant.To compare the detorque values between the study groups, analysis of variance, or its non-parametric equivalent, the Kruskal-Wallis test, was used. SPSS 17 was used for data analysis.  The distribution of the variables in each group was analyzed by the Kolmogorov-Smirnov test, and because of their normal distribution, a parametric test, such as ANOVA, was used to compare them. Post hoc Tukey tests were used for two-by-two comparisons of the groups.P < 0.05). In addition, there was a significant difference between the salivary and chlorhexidine groups and between the tetracycline and chlorhexidine groups (P < 0.05).According to the Tukey test results in  Incorrect torque when tightening the screw is an influential factor in screw loosening and breakage and other technical problems with implant systems. One of the causes of unintentional loosening of screws is insufficient torque, and one of the causes of screw fractures is excessive torque.One of the problems with implant prostheses is the loosening of the screw that connects different parts. Unstable and loose prosthetic screws can lead to more serious complications such as screw fracture, prosthesis loosening, or implant failure.When exerted on natural teeth, functional and parafunctional forces create a physiological adjustment in the periodontal tissue, but occlusal trauma can result when this force is greater than adaptive capacities. Unlike natural teeth, excessive occlusal forces due to the lack of periodontal ligamentaround the implant fixtures can cause mechanical problems such as screw loosening, screw failure, and even fixture failure. The most important and common implant problems include implant failure, loosening, infection, inflammation, bone and tissue failure, damage to adjacent structures, etc. Inflammatory processes around implants are relatively similar to those around natural teeth, except that infections around implants cause more destruction, mainly due to the absence of periodontal ligaments. The gap between the two components causes bacterial proliferation, inflammation, and bone loss around the implant. Bacterial biofilms around the implant‒abutment complex affect the biological width, compromising the bone margin. Finally, the soft tissue margin recedes, which affects the aesthetic outcome.Controlling inflammation around dental implants is critical to reducing the rate of bone resorption in the crestal region, the health of the surrounding soft tissue, and increasing the efficiency and life of dental implants. The current study examined the effect of two different antibacterial agents used to control microbial growth and reverse torque values of the abutment screw. In this study, the reverse torque with artificial saliva was 23.37 N/cm2, which was higher than the other two groups of tetracycline and chlorhexidine. ANOVA and Tukey tests revealed that this difference was significant.A major challenge is preventing bacterial proliferation at the implant‒abutment connection to minimize inflammatory reactions and maximize bone stability in the crestal area. leading to inflammation at the implant‒abutment connection and reducing implant durability. Microleakage at the implant‒abutment connection is the most important cause of the inflammatory reaction around the implant. Microleakage causes bacterial colonization around the implant‒abutment complex, leading to the onset of the pathophysiological process of bone loss, followed by implant loss.Not using antibacterial agents increases the accumulation of bacteria and leakage into implant cavities,2. Chlorhexidine reduces the accumulation of bacteria, toxins, and bacterial leakage into the implant cavity, and according to the current study, it reduces the level of the detorque values. These antibacterial agents in the implant cavity can lead to slippery surfaces and reduce the detorque values of abutment screws.When chlorhexidine was used in the second group of this study, the reverse torque reached 20.02 N/cm reported that 0.2% chlorhexidine gel on the implant cavity reduced the detorque and preload values and increased the risk of screw loosening, consistent with the present study. Paolantonio et al showed that using 1% chlorhexidine gel in the implant cavity before placing the abutment significantly decreased bacterial colonization in the implant cavity over six months. The detorque value in the chlorhexidine group was similar to that in a study by Micarelli et al. They also stated that contamination reduced the level of detorque. Since contamination was unavoidable in a laboratory process, they suggested using plasma argon cleaners to decontaminate the screw‒abutment complex.Asli et al2, which was significantly lower than in the second group. Park et al also showed that the detorque value in the tetracycline group decreased due to the increased slipperiness. The greater reduction in this compared to the chlorhexidine group was attributed to the nature of the antibacterial agents. Chlorhexidine is water-based, and tetracycline is oil-based, and the lubricity of tetracycline is higher than that of chlorhexidine. Therefore, the reduction in detorque values was greater with tetracycline. The presence of a lubricant and its type in the screw‒abutment complex can reduce the coefficient of friction, depending on the type of lubricant. Since both chlorhexidine and tetracycline reduced reverse torque values in this study, these two substances are not considered suitable lubricants.Tetracycline decreased the detorque value in the third group in this study, which was greater than in the second group. The detorque value was 16.25 N/cm The present study was conducted by taking into account some hypotheses and had some limitations. To improve and expand the topic, the following should be investigated: the bacterial accumulation around the abutment, the effect of antibacterial lubricants, and the effect of various impurities and cleaners on detorque values. According to the present study and other studies, using antibacterial agents reduces the detorque values of abutment screws. The reduction in detorque with oil-based antibacterial agents is more than that with water-based ones. The authors would like to thank the Prosthodontics Department, Faculty of Dentistry, Tabriz University of Medical Sciences, for their cooperation. No funding was requested for this study. The study protocol was approved by the Ethics Committee of Tabriz University of Medical Sciences under the code IR.TBZMED.VCR.REC.1398.438. The authors declare no competing interests with regard to the authorship and/or publication of this article.


Detection of allergen-specific immunoglobulin E (IgE) antibodies (Abs) in serum would allow for screening of the causative allergen in patients with type-I allergy. In this study, we developed a new assay method to detect allergen-specific IgE Abs, which involved crosslinking the plural FcεRIα molecules with an allergen and detection using an amplified luminescence proximity homogeneous assay (AlphaCL). First, the allergen concentration, bead concentrations, and incubation time were optimized for the detection of anti-2,4-dinitrophenyl (DNP) IgE Abs in buffer. Under optimal conditions, AlphaCL was able to detect DNP-specific IgE Abs in simulated human serum at levels comparable to those in serum from type-I allergic patients. When AlphaCL was used to detect anti-DNP IgE Abs, no signal counts were obtained with the monovalent allergen 2,4-dinitrophenylated poly-γ-glutamic acid, whereas high signal counts were obtained with the multivalent allergen DNP-BSA. This confirmed that AlphaCL could specifically detect allergen-specific IgE Abs with the ability to crosslink a multivalent allergen. In summary, we have established a new assay model using AlphaCL to detect allergen-specific IgE Abs with FcεRIα crosslinking ability in human serum. This simple and practical assay model may be applied as a new diagnostic tool for patients with type-I allergy. The detection of allergen-specific IgE Abs in serum could be applied to screen for the causative allergen in patients with type-I allergy represented by food allergy6. In clinical practice, fluorescence enzyme immunoassays (FEIAs), such as ImmunoCAP™, are widely used to measure the serum levels of allergen-specific IgE Abs9. However, the FEIA system detects IgE Abs regardless of their ability to crosslink the plural FcεRIα with an allergen, resulting in false-positive diagnoses11. The histamine release test (HRT) and basophil activation test (BAT) using basophils from the peripheral blood of a patient are performed to detect IgE Abs with the ability to crosslink13. However, the differences in basophils condition after blood sampling can affect the results14. In addition, HRT and BAT cannot be applied to patients with basophils that are unreactive or show low reactivity to the anti-IgE Abs used as positive controls15. To improve these tests, the IgE crosslinking-induced luciferase expression (EXiLE) test using cultured mast cells was established for the detection of allergen-specific IgE Abs17. However, the EXiLE test is complicated in terms of cell management and manipulation, and a limited number of clinical facilities can perform this test. In the case of food allergy, a false-positive diagnosis may decrease the quality-of-life of a patient because of an unnecessary elimination diet, and a false-negative diagnosis could lead to anaphylaxis if the causative allergen in food is ingested20. Therefore, a highly sensitive and accurate assay method for the diagnosis of type-I allergy is needed in clinical practice.Type-I allergic symptoms are elicited by crosslinking of plural allergen-specific immunoglobulin E (IgE) antibodies (Abs) bound to high-affinity IgE receptor alpha subunits (FcεRIα) on the surface of mast cells and basophils with an allergen21. Alpha technology is a bead-based proximity assay, which is used to detect the molecular interactions among biomolecules22.Recently, we established a new assay, the amplified luminescence proximity homogeneous assay (AlphaCL), to detect functional autoantibodies against FcεRIα and IgE Abs, which can crosslink the plural FcεRIα molecules and IgE Abs on the surface of mast cells and basophils present in sera, in patients with autoimmune chronic spontaneous urticaria (aiCSU)In the current study, we developed a new assay method based on AlphaCL to detect allergen-specific IgE Abs, which can crosslink the plural FcεRIα molecules with an allergen. The principle of this assay model imitates mast cell activation by crosslinking of allergen-specific IgE Abs bound to FcεRIα on the surface of the beads with an allergen. As shown in Fig. This new assay model differs from our previous ones in the process of cross-link formation between acceptor and donor beads. When detecting the functional IgG autoantibodies against IgE Abs and FcεRIα in sera from aiCSU patients, the autoantibodies bind directly to each protein conjugated on both beads and can cross-link beads to each other. In contrast, when detecting the functional IgE Abs specific to an allergen in sera from patients with allergy, the allergen-specific IgE Abs must bind to the FcεRIα protein on the beads. Therefore, tripartite complex must be formed between the allergen, the allergen-specific IgE Abs and the FcεRIα conjugated on beads. In addition, a higher sensitivity is required to detect allergen-specific IgE Abs than IgG autoantibodies because the amount of IgE Abs in the serum may be much lower than that of IgG Abs. Furthermore, it is important to consider monovalent allergens. In this study, we focused on these issues and established optimal detection model for allergen-specific IgE Abs.To optimize the detection of anti-DNP IgE Abs in immunoassay buffer, we evaluated the appropriate concentrations of DNP-BSA and beads, and the incubation time. DNP-BSA gradually increased the signal counts up to 0.1 μg/mL, and then decreased the signal counts at concentrations of 0.00625–1.6 µg/mL . In the buffer solution, the signal counts logarithmically increased depending on the concentrations of anti-DNP IgE Abs up to 0.8 μg/mL, and then reached a plateau Fig. A. In theTo evaluate whether AlphaCL can specifically detect the allergen-specific IgE Abs with the ability to crosslink a multivalent allergen, we attempted to compare the differences in the detection of anti-DNP IgE Abs against monovalent allergen and multivalent allergen by ELISA and an AlphaCL assay. We used 2,4-dinitrophenylated poly-γ-glutamic acid (DNP-γPGA) as a monovalent allergen. The absorbance values increased in the presence of DNP-γPGA or DNP-BSA compared with unconjugated γPGA or BSA  the use of non-basophil cells or cultured mast cells, (2) the highly specific detection of functional allergen-specific IgE Abs, (3) the feasibility of the assay in any facility, (4) the minimally invasive nature of the assay for subjects, and (5) the fact that it is substitutable for various allergens.Allergen-specific IgE Ab detection can be utilized to screen for the causative allergen in patients with type-I allergyTo optimize the detection for anti-DNP IgE Abs, we first determined the appropriate concentrations of DNP-BSA and beads, and the incubation time. DNP-BSA increased the signal count gradually up to 0.1 μg/mL and then decreased Fig. A. The de27. Therefore, high concentration of anti-BSA IgG Abs in human serum might disrupt the bindings of anti-DNP IgE Abs with DNP-BSA in this assay model. In addition, we speculate that the detection of anti-DNP IgE Abs may be disrupted by the highly abundant serum components such as non-specific free IgE and IgG Abs as well as anti-BSA IgG Abs. In addition, the serum components with free-radical scavenging capacity may inhibit the transfer of singlet oxygen from the donor beads to the acceptor beads28. The concentrations of allergen-specific IgE Abs were reported to be ~ 0.00084–0.24 μg/mL in sera from patients with type-I allergy29. Therefore, it may be necessary to remove the interfering components including IgG Abs in the serum to improve the sensitivity of our assay.Next, we detected DNP-specific IgE Abs in simulated human serum using AlphaCL. The signal counts in the buffer increased up to 0.8 μg/mL of anti-DNP IgE Abs, and then reached a plateau Fig. A. This r30. As mentioned above, ~ 30 DNP groups conjugated to one BSA molecule used in this study. In contrast, one γPGA molecule can be conjugated with only one DNP group because this protein has one amino group at the N-terminus. The higher absorbance values in ELISA were obtained in the presence of DNP groups compared to their absence . Further studies are necessary to confirm whether the levels of allergen-specific IgE Abs in sera from patients with type-I allergy can be determined by AlphaCL.The limitation of this study was that we used a simulated serum sample prepared from pooled healthy subjects instead of serum from patients with type-I allergy. Previous reports have shown that patients with type-I allergy possess allergen-specific IgG Abs, in addition to allergen-specific IgE AbsIn conclusion, we established a new assay method using AlphaCL to detect allergen-specific IgE Abs with FcεRIα crosslinking ability in serum. This simple and practical assay method could be applied to detect various allergen-specific IgE Abs and has potential as a new diagnostic tool for patients with type-I allergy.22. The levels of allergen-specific IgE Abs can be quantitatively determined by measuring the intensity of emission peak signals using a multimode plate reader.To construct our new assay model, we used DNP-BSA  as an allergen and mouse anti-DNP IgE Abs  as allergen-specific IgE Abs. When donor beads come into proximity by the crosslinking of allergen-specific IgE Abs bound to FcεRIα conjugated on the beads with an allergen, the acceptor beads can receive the singlet oxygen that is released from the donor beads by excitation at 680 nm. The energy generated by singlet oxygen in the acceptor beads results in a sharp emission peak at 615 nm . For evaluation of the bead concentrations, 20 µL of DNP-BSA (0.1 µg/mL) and 20 µL of anti-DNP IgE Abs (0.2 µg/mL) were incubated in the plate for 1 h at RT. Then, 10 µL of the bead mixture at different concentrations  were added and incubated in the same plate for 24 h at RT in the dark. For the incubation time, 20 µL of DNP-BSA (0.1 µg/mL) and 20 µL of anti-DNP IgE Abs (0.2 µg/mL) were incubated in the plate for 1 h at RT. Then, 10 µL of the bead mixture  were incubated in the same plate for 1–48 h at RT in the dark. The Alpha signal counts were measured using a multimode plate reader , and calculated as net counts by subtracting the counts in buffer containing no anti-DNP IgE Abs from the total counts in each sample. All experiments were performed in duplicate.To detect anti-DNP IgE Abs, we also prepared donor and acceptor beads  conjugated with FcεRIα proteins  according to the manufacturer’s protocol (PerkinElmer). The beads were diluted with immunoassay buffer (PerkinElmer) and mixed at appropriate concentrations. To optimize anti-DNP IgE Ab detection, we evaluated the allergen and bead concentrations, and the incubation time. To determine the DNP-BSA concentration, 20 µL of DNP-BSA (0.00625–1.6 µg/mL) and 20 µL of anti-DNP IgE Abs (0.2 µg/mL) were added to a 1/2 AreaPlate-96 plate (PerkinElmer) and incubated for 1 h at room temperature (RT). Then, 10 µL of the bead mixture  was incubated in the same plate for 24 h at RT in the dark. Our previous report showed that 24 h incubation was optimal to detect the anti-IgE autoantibodiesTo detect DNP-specific IgE Abs in human serum using AlphaCL, we prepared simulated human serum by adding anti-DNP IgE Abs into commercially available human pooled serum (Sigma-Aldrich). DNP-BSA (0.1 µg/mL) was incubated with an equivalent volume of buffer or the simulated serum sample containing anti-DNP IgE Abs at concentrations of 0.1–2.0 µg/mL for 1 h, and then 10 µL of the bead mixture  was incubated in the plate for 24 h. The signal counts were measured and calculated according to the method described above. All experiments were performed in duplicate.33 with a slight modification. Briefly, 1-fluoro-2,4-dinitrobenzene  dissolved in 500 µL of ethanol was slowly mixed with γPGA sodium salt  dissolved in 500 µL of 1.2 M sodium hydrogen carbonate solution. After incubation for 2 h at RT, the solution was centrifuged at 3000×g for 30 min using Centricut® Mini V-10 . Then, the pellet was collected by dissolving with ultrapure water and acidified to pH 7.0 with diluted hydrochloric acid.To demonstrate that AlphaCL can only detect allergen-specific IgE Abs to multivalent allergens with at least two or more IgE-binding sites, we prepared DNP-γPGA as a monovalent allergen, according to the method described by Sanger et al.34 with a slight modification. Briefly, F8 MaxiSorp loose Nunc-Immuno Modules  were coated with 100 μL of DNP-conjugated or unconjugated γPGA (1 mg/mL), or BSA , dissolved in phosphate-buffered saline  overnight at 4 °C. After washing with PBS containing 0.05% Tween-20 (PBS-T) three times, the plates were incubated with 1% Block Ace®  for 2 h at 37 °C. After washing with PBS-T, 100 μL of anti-DNP IgE Abs dissolved in 1% Block Ace® was applied and incubated for 2 h at 37 °C. The wells were washed with PBS-T, and then incubated with 100 μL of horseradish peroxidase-conjugated goat anti-mouse IgE Abs  (diluted 1:7000 with 1% Block Ace® solution) for 1 h at RT. The wells were washed with PBS-T and incubated with 100 μL of 3,3′,5,5′-tetramethylbenzidine solution  at RT. After a 15-min incubation, the reaction was terminated with 100 μL of 1 M phosphoric acid. Absorbance was determined at 450 nm against 620 nm as a reference using a Multiskan GO™ (Thermo Fisher Scientific).ELISA was performed according to the method described by Yamada et al.For the AlphaCL assay, DNP-conjugated or unconjugated γPGA  or BSA  was incubated with 20 µL of the simulated serum sample containing anti-DNP IgE Abs (0.2 µg/mL) in the plate for 1 h at RT. Then, 10 µL of the bead mixture  was incubated in the plate for 24 h at RT in the dark. Measurements were performed using NIVO™. The signal counts were measured and calculated according to the method described above. All experiments were performed in duplicate.P < 0.05 was considered statistically significant.Data were displayed as the means ± standard deviations of the mean (S.D.), and the strength of the correlation in the results between anti-DNP IgE Abs concentration and Alpha signals were analyzed by Spearman’s correlation coefficient by rank test. Mean value differences between DNP-labeled and unlabeled protein were assessed using Student’s t-tests.