Ice accretion oneconomically valuable and strategically importantsurfaces poses significant challenges. Current anti-/de-icing techniquesoften have critical issues regarding their efficiency, convenience,long-term stability, or sustainability. As an emerging ice mitigationstrategy, the thin-film surface acoustic wave (SAW) has great potentialsdue to its high energy efficiency and effective integration on structuralsurfaces. However, anti-/de-icing processes activated by SAWs involvecomplex interfacial evolution and phase changes, and it is crucialto understand the nature of dynamic solid–liquid–vaporphase changes and ice nucleation, growth, and melting events underSAW agitation. In this study, we systematically investigated the accretionand removal of porous rime ice from structural surfaces activatedby SAWs. We found that icing and de-icing processes are strongly linkedwith the dynamical interfacial phase and structure changes of rimeice under SAW activation and the acousto-thermally induced localizedheating that facilitate the melting of ice crystals. Subsequently,interactions of SAWs with the formed thin water layer at the ice/structureinterface result in significant streaming effects that lead to furtherdamage and melting of ice, liquid pumping, jetting, or nebulization. Comparedto glaze ice, the formation of rime ice is mainly regarded as an instantsurface freezing process of supercooled water droplets, with featuressuch as low liquid water contents , strong supercooling/low temperatures , and porous  and cluster shapes after freezing.6 Theformation of porous structures is mainly due to the quick freezingof the supercooled droplets without residual liquid water that fillsthe gaps. Under low-humidity conditions and at subzero temperatures,supercooled droplets are easily deposited on the structural surfacesand then gradually form a thick layer of rime ice.7Ice accretion on structuralsurfaces is one of the critical hazardsin aerospace, power transmission, offshore platform, and wind turbinesectors.8) and active techniques . However, their efficiency and sustainability for ice protectionhave significant limitations. For example, icephobic surfaces oftenhave issues of poor mechanical or long-term durability.14 The chemical fluids used for the removal of accreted ice could causesevere environmental issues.16 The electrical heatingmethod often consumes excessive energy for ice prevention or removal.Therefore, innovative ice mitigating techniques with high energy efficiencyand environment-friendly features are critically required.Commonly applied ice mitigation technologies include both passiveapproaches  and their hybrid waves can be generatedand then propagate along the structural surfaces.23 Compared to the conventional bulk piezoelectric material-based SAWdevices, thin-film-based SAWs have the advantage that they can integratemultiple functions into a single structure on different substrates,such as silicon, metals, glass, or polymers.24 Besides the wide applicability on various substrate materials, SAWscan be generated and then propagate on surfaces with different features,even on flexible and bendable surfaces if thin film technology isused.25 Furthermore, the direction of propagatingSAWs can be designed across the whole solid surfaces.26 Acoustofluidic phenomena generated using thin-film SAWdevices, including liquid mixing, transportation, jetting, nebulization,droplet generation, and particle/biological cell sorting and manipulations,have recently been reported.30Surfaceacoustic wave (SAW) technologies have been widely appliedin wireless communications, acoustofluidics, sensors, particle/cellconcentrating, and micro-heaters.32 However, interfacial behaviors, ice removal, and prevention mechanismsfor both anti-icing and de-icing of the rime ice under propagatingSAWs have never been explored. Compared with conventional acoustofluidicresearch of sensors or laboratory-on-a-chip using the thin-film SAWdevices,34 ice protection and mitigation using thin-film SAWsare more complex, mainly because there is varied phase evolution  and dynamic evolutionof interfacial microstructures during the processes under the agitationof propagating waves. There is a lack of in-depth investigations onthe interfacial responses and phase evolution driven by SAWs duringthe icing and de-icing processes, which restricts the further explorationof SAW devices for ice mitigation.In the field of ice mitigation, thin-film-basedSAWs have alreadybeen demonstrated to effectively generate both acoustic wave vibrationand thermal effects on the device surface, thus offering great potentialsfor both anti-icing and de-icing with a high efficiency.This study is focused onthe anti-icing and de-icing mechanismsof porous rime ice on a structural surface , integratedwith ZnO-based thin-film SAWs. We first investigate the fundamentalissues about interactions of SAWs with the rime ice and/or liquid/icemixtures. Then, we focus on the experimental studies of anti/de-icingperformance for rime ice using thin-film SAW devices. Finally, theevolution of ice morphology and phase changes at different humiditylevels under icing conditions and different SAW powers are investigated,from which the de-icing and anti-icing mechanisms using thin-filmSAWs are verified.1.131 They play key roles in the anti/de-icing processby preventing ice accumulation and effectively removing the formedice.31 In this section, we address thisissue by considering the phase evolution of ice, water, and vaporand connect them with anti/de-icing mechanisms with SAWs. The hypothesesabout the anti/de-icing processes and mechanisms are first establishedand then proved by the designed experiments. The whole process canbe considered in three different configurations with the presenceof  and the acoustic thermal heatingeffect are two main mechanisms for ice protection using SAW devices.1.21.2.139 Most of these models were based on two assumptions,that is, the acoustic waves have relatively low frequencies (fromhundred to thousand Hz); and the rime ice is a rigid-frame model witha stable structure. However, the structure of real rime ice is highlyporous and fragile. To ascertain the transmission of SAW energy fromthe substrate to the porous rime ice, we use the Johnson–Champoux–Allard(JCA) fluid (such as air) model to determine the acoustic impedanceof the porous layer (Zp)44a∞ andϕ are the tortuosity and porosity of the porouslayer, respectively, η and ρ0 are the viscosityand density of the fluid  in the porous layer, respectively,ω is the angular frequency of the wave, Pr isthe Prandtl number, σ is the flow resistivity of the porouslayer, Λ and Λ′ are the viscous characteristiclength and the thermal characteristic length of the porous layer,respectively γ0 is the ratio of specific heats, and P0 is the static pressure.At the first stage, whereonly solid and porous rime ice crystalsexist, the key phenomena at the interface are the propagation of SAWson the device’s surface, mainly through the interface betweenthe device and the porous rime ice. We believe that the acoustic impedanceof the porous rime ice is a key parameter that affects the propagationof SAWs along the surface of the structure. Currently, there are differenttheoretical models (such as Biot’s theory and Pyett’stheory) proposed to investigate the acoustic impedance of snow orrime ice with various densities.45 reported that there is a large impedance mismatch between the surfaceof the SAW devices along with the wave propagation and the poroussuperhydrophobic nanoparticle layer coating (ϕ ≈ 0.57),which results in a wave reflection coefficient of 0.998, calculatedusingBased on theabove background information, Biroun et al.This low wave reflection coefficient indicates a weak transmissionof acoustic energy at the substrate–porous layer interface.Although it is challenging to quantify the porosity of the rime icelayer, we can reasonably assume that the porosity of the rime icewill be higher than that of the nanoparticle porous coating in ref . Therefo24 The heating effect then conducts the viscousdissipation of the acoustic energy into the liquid such as a sessiledroplet, as extensively reported in refs20 and46 –474849.Apart from the significant surface vibrations, the applied radiofrequency power also induces a localized heating effect  in the thin-film SAW devices due to theSAW energy dissipation. This thermal effect is generated by the high-frequency-inducedmechanical vibration and stress generated in the substrate.1.2.2Fs,which is given using the following expression521 = jϑ(with j2 = −1) is theattenuation constant and L and υS are the wave velocities in the liquid and on the solid surface,respectively. A is the wave amplitude, ω isthe angular velocity, k is the wave number of theleaky SAW, and subscripts i and j = 1, 2, and 3 represent the x, y, and z coordinates. Sudeepthi et al.53 reported the transition of wetting behaviorson a porous and nanoparticulate surface with the application of SAWs.They observed that with the agitation of SAWs, the state of surfacedroplets was changed from a Cassie state to a Wenzel state, indicatingthat SAW agitations caused the surface water to penetrate into theporous layer. Therefore, this indicates that the SAW streaming forcecan effectively prompt water penetration and propagation into theporous rime ice layer and also significantly enhance the acousticthermal effect.With the activation of SAW energy, the interfacialice layer is partially melted, thus forming the melted liquid at theinterface due to the phase evolution. Once the liquid phase startsto appear at the device/ice interface, the de-icing front at the interfacesee 1 willbeFR) and acoustic streaming drag force (Fd), which are56p0 is theacoustic pressure, Vp is the volume ofthe ice crystals, β and ρ are the compressibility anddensity of the melted liquid and ice crystals, respectively, φis the acoustic contrast factor, λ is the wavelength of theacoustic waves, x is the distance from the pressurenode, and μ, r, and v arethe dynamic viscosity of the liquid, radius, and relative velocityof the ice crystals, respectively. The significant flowing of icecrystals can accelerate their elimination and enhance the mass changesinside the liquid.Under the activation of propagating SAWs, thisde-icing front quickly moves along the SAW propagating direction,away from the IDT area. With the continued de-icing process, the porousrime ice will shrink gradually until it is changed into multiple independentice crystals flowing with the internal streaming induced by SAWs.In this case, the semi-melted ice crystals dispersed in the liquidare governed by the acoustic radiation force , someice crystals may also be initiated in the gas phase from the watervapor. Thus, in this study, both supercooled droplets (the main phase)and ice crystals (the secondary phase) will be discussed in the anti-icingprocess.Based on the theory of homogeneous ice nucleationin the air, supercooleddroplets would be the main phase that flies and is attached to thesolid surface and then nucleates, grows, and forms the porous rimeice due to the low temperature nucleation centres.rc, and increasing the critical free energy of heterogeneous ice nucleation,ΔG*, which has been reported in ref . The crystalline structure of the ZnO film was analyzed usingan X-ray diffractometer  with Ni-filtered CuKα radiation . TheXRD pattern of the ZnO film on the aluminum substrate shows a strongpeak of the ZnO (0002) diffraction plane. This indicates the c-axis preferential growth of the Wurtzite-structure ZnOfilm. The interdigital transducers (IDTs) were patterned on top ofthe ZnO thin film using a conventional photolithography and lift-offprocess. A bilayer of Cr/Au with a thicknesses of 20/100 nm was preparedusing a thermal evaporator (EDWARDS AUTO306) as the electrode. TheIDTs were designed with a wavelength of 400 μm, comprising 30pairs of electrodes. The corresponding Rayleigh wave frequency measuredusing a network analyzer was 7.22 MHz.A ZnOfilm of ∼5 μm thickness was deposited on 1.5 mm-thickAl plates using the DC magnetic sputtering technique. A zinc targetwith 99.99% purity was used during the deposition. The DC power was400 W, and the Ar/O2.2To createa stable and constant icing/anti-icing environment, the experimentwas conducted in a freezing chamber that was built based on a coldplate with a sealed resin shield. The accurate humidity was achieved usingan atomizer (Omron Ultrasonic Nebulizer NE-U17) that generated wateraerosols with controlled vaporizing power and imputing speed. Thevelocity of airflow (∼3.8 m/s) and temperature consistencyinside the chamber was stabilized using an electric fan. The coldplate was set at −6.5 °C, which kept the environmentaltemperature at −1 °C and the device temperature at −10°C. Before the start of icing, the SAW device was cooled downin the chamber for 20 min in advance. Then, the icing process wascarried out with RH levels of 60, 70, 80%, and 90%, respectively.The anti-icing study was performed with different RH levels and SAWpowers (from 0.002 to 2.300 W). The icing duration lasted for 20 minwhile an IDS camera with a Navitar 12× objective lens was usedto record the ice morphology from the top and side of the device.The mass of rime ice accumulated was measured after the icing process.Each test was repeated three times to get the average value. An infraredcamera was used to monitor temperature changes of the SAW device surfacewith a humidity of 25% in the same chamber with the same airflow speed.2.311 was measured every 1 min.After the ice formation, the SAW was applied with various powers (from0.400 to 2.300 W with proper gaps) to evaluate the de-icing performance.A high-speed camera (HotShot 1280 CC) with a Navitar 6.0× zoomlens and a 1.5× objective lens and the IDS camera with a Navitar12× objective lens were also used to record the de-icing processfrom the top and side view.To do the de-icingtests, all the samples were cooled down in the chamber with a temperatureof −6.5 °C for 20 min. The de-icing was carried out (for5 min at most) with various RH levels of 60, 70, 80, and 90%, respectively.The SAW reflection signal of S33.1k2) of the SAW devicewas ∼1.75%, whereas the temperature coefficient of frequency(TCF)24 was ∼248 ppm/°C (seethe Supporting Information).The SAW deviceused in this study was formed on the ZnO thin film deposited on a1.5 mm aluminum plate, and the designed wavelength was 400 μmwith a measured Rayleigh resonant frequency of 7.22 MHz. The electromechanicalcoupling coefficient  are shown in Figure S1 inthe Supporting Information The resultsshow that the ice accretion caused serious damping of SAW signals.All the necessary basic information about the SAW device used in thisstudy is summarized in Thereflection spectra SSupporting Information. Selected infrared imagesare shown in Figure S3 in the Supporting Information. As expected, with the increase in the applied SAW power, the surfacetemperature increases. When the SAW power is applied continuously,the recorded temperature changes at a substrate temperature of −10°C become more significant than those of the substrate at roomtemperature .The obtained data are summarized in the Supporting Information.The thermal heating effect on the surface of the SAWdevice wascharacterized using an infrared camera. The obtained temperature changesof the SAW device surface with various SAW powers within 90 s in anambient environment (17 °C) and in a cold chamber (−10°C) with 25% humidity are summarized in Figure S2 in the 3.259 For the possibly attached ice crystals, the surface vibration inducedby the acoustic wave agitates the attached areas of these ice crystals,which destroys their structures. At the ice/device interface, theacoustic thermal effect causes a local heating effect; thus, the icecrystals melt easily. The formation of a liquid layer provides a goodmedium to absorb SAWs, leading to the internal flow inside the liquid,as explained in Based on the above results and discussion in 3.3Toperform de-icing tests, the rime ice was first formed at various RHlevels for 20 min, and then, the SAW power was applied to study thede-icing phenomena. P is the SAW power,τ is the consumed time of removing the ice on the IDT area,and m is the mass of ice on the IDT area. 61 As it is well known, the conventionallyused electrothermal de-icing technique generally has low energy efficiencyand high energy consumption,62 whereas the SAW technologyhas its advantages such as the high efficiency for the de-icing processbecause of the combined effects of acoustic vibration and acousticthermal effects, both of which are localized at the ice/device interface,as reported in Yang et al.31Based on the obtained results, the specific energy consumptionfor removing ice can be estimated using the following expressionBased on the experimental observations, the de-icing process canbe summarized in The first stage in The second stage in 24 which we haveobserved using the high-speed camera. These two effects lead to thefaster conduction of the thermal effect than that of acoustic wavesin the area away from the IDT region. The mixed front with ice andwater had been quickly moved along the surface until all the ice disappeared,with the remaining liquid vibrating, nebulizing, or evaporating onthe device’s surface (see the videos about the de-icing processin the Supporting Information).With the further de-icing process, the third stage is formed asshown in With further SAW agitation, phase changes happen, starting nearthe top area around the IDT. An icing front consisting of a liquid/icemixture is formed and quickly moves along the SAW direction underthe hybrid action of the acoustic wave and thermal heating effects.The width of the de-icing front is enlarged obviously with the continuationof the de-icing process from 6c. The dIn brief, we have confirmed that thekey de-icing mechanisms arethe phase changes induced by the hybrid effect of acoustic waves andthe thermal effect. The surface vibrations induced by the acousticwaves affect the interfacial structures between the rime ice and thedevice, leading to the breakup and collapse of ice crystals or clusters.With the accumulation of the thermal effect, phase change also occursat the ice/device interface, and the melted liquid and the ice crystalsare quickly merged. Apart from the above two major effects, when theSAW waves propagate into this ice–liquid mixture front, theinner flow and streaming force significantly enhance the exchangeof mass and heat, which effectively promotes the de-icing process.4In this study, anti-/de-icingmechanisms of rime ice using thin-filmSAW technology were studied systematically. Anti-icing results showedthat the anti-icing performance was improved significantly with theincrease in the SAW power, and de-icing results showed that the de-icingenergy efficiency was quite high for the SAW device, even in a severelyfrozen environment at a high humidity level. On comparing with thepotential thermal energy consumption of thermal melting ice, bothacoustic wave vibration and acoustic heating play key roles duringthe de-icing process. The surface vibration induced the breakup andcollapse of ice crystals or clusters. The accumulation of heat promptedthe melted liquid that absorbed the SAW waves and led to the internalstreaming to enhance the exchange of mass and heat. These effectsgenerated further ice melting, liquid pumping, jetting or nebulization,and evaporation during the icing process, while the formation andmovement of the de-icing front in the de-icing process were enhancedaccordingly.


The occupation of the site appears to have been influenced by changes in the local climate and by extreme El Niño events. Our results highlight the great adaptability of these human groups over the span of a millennium and their capacity to react to the climatic changes and hazards that characterise this region.Long considered on the margins, far from the major cultural traditions, the Sechura Desert is situated at the crossroads between the cultures of southern Ecuador and those of the northern Peruvian coast and preserves a large number of varied archaeological sites. Despite this evidence, little is known about the societies that inhabited this region during the Holocene. Exposed to natural hazards, including El Niño events, and to major climatic changes, they were able to adapt and exploit the scarce resources that this extreme environment offered them. Because of this rich history, we have been conducting archaeological research in this region since 2012 in order to clarify the dynamics of human occupation and their links with climate oscillations and environmental changes. This paper present the results of a multidisciplinary study of Huaca Grande, a mound located on Nunura Bay, 300 m from the Pacific Ocean. The nature of the human occupations at Huaca Grande was varied, and several adjustments occurred over time. The subsistence economy was based mainly on local marine resources and a continual use of terrestrial vegetal resources. However, a major change occurred in the more recent occupations, with the apparition of non-local resources (maize and cotton) indicating that Huaca Grande was connected to trade networks. The results show two main phases of occupation separated by a long abandonment (mid-5th century CE to mid-7 Despite difficult environmental and climatic conditions, the Sechura Desert has been occupied by human groups for at least 7000 years (from 5590 ± 90 RCY BC) –3. Humancostero, or COA EN] of 2017; [The coastal environments of northern Peru are particularly sensitive to climatic oscillations and extreme events caused by El Niño . These of 2017; , 11, theof 2017; –15. SuchHence, in this as yet poorly known archaeological and palaeoenvironmental context, for which archaeologists have only fragmentary and incomplete data, we chose to study the Huaca Grande archaeological site  because We started new excavations there in 2015. Our goal was to achieve a better understanding of human settlement on Nunura Bay  and of tIn order to be able to draw the maximum amount of information from the exceptional archaeological and sedimentary sequence of Huaca Grande, we developed an intra-site multidisciplinary integrated approach that includes geoarchaeological and bioarchaeological studies. This approach, which is still little applied in Peru, is mainly based on detailed and comprehensive chronostratigraphic analyses of the archaeological sequence , in combination with the study of the faunal and botanical remains  and radiocarbon dating, in order to (i) establish the age and duration of the occupations; (ii) identify the function of the site and the activities that took place there; and (iii) understand the subsistence economies adopted by human groups through the identification of the resources they exploited.The Sechura Desert is a vast coastal region in northern Peru (lat. 6°S–7°S), situated between the Piura and Lambayeque valleys , bordereclimate-data.org).Weather conditions are fairly stable throughout the year. The average temperature is 23.9°C, and the temperature variation between the warmest month  and the coldest month  is about 6°C. The very rare rainfall is concentrated between late summer and early autumn, with a peak of 111 mm on average in March , which became filled with water during the more humid climatic phases throughout the Holocene and during El Niño events up to the present day. The two main ones are the Quebrada Verdun, which crosses the central part of the land bordering the bay, running southeast to northwest, and the Quebrada Nunura, which runs along the margins of the Huaca Grande mound and has its mouth to the south of the mound . These initial data, in addition to the radiocarbon dating results, encouraged us to resume the excavations on this mound in order to better understand the activities that took place there and to place them in the regional chronological sequence. To do this, we dug two 2 m × 2 m test pits (A4.1 and A4.2)–of which the former was eventually expanded to a 10 m × 5 m area–as well as a 10 m × 5 m excavation area (A4.3). The objective of the latter was to relocate the trench excavated in the 1970s.During the field campaign by Cárdenas et al. , 2 threeAll the archaeological remains and samples collected during the excavation campaigns are currently stored at the Ministry of Culture of Peru, except for the micromorphological thin sections and the charcoal samples, which are stored at the Malher Centre  and the MSH Mondes  respectively.Study of the Huaca Grande stratigraphic sequence has required the implementation of a specific methodology adapted to the excavation conditions and the variety of archaeological remains recorded. Stratigraphic profiles 1  and 4  of area A4.1 were systematically described, sampled and studied. Samples of sediment for sedimentological analyses were taken from each layer . In addi14C dating, spanning the entire archaeological sequence, were collected from the stratigraphic profiles and from the camelid deposit found during the excavations ; 1 by Beta Analytic (USA); and 2 (the camelid collagen) by AMS ECHoMicadas , after chemical extraction [Thirteen samples for traction  at the rAll dates were calibrated at the 2σ age range (≥95%) using OxCal 4.4  and the The good state of preservation and the representativeness of the excavated skeletons allowed a biological identification of the two individuals buried at Huaca Grande. The estimation of the age at death of these two adult individuals is based on the study of their dentition  and the Regarding sexual diagnosis, the good preservation of the coxal bone of both adult individuals allows the use of two reliable methods based on a world reference sample. The first method is morphoscopic with a minimum reliability of 95% . It consThe Huaca Grande faunal remains are comprised of mammals , birds, fish, molluscs and other marine invertebrates. Each layer of the stratigraphic profile (0–2.80 m) was systematically sampled, and the sediments were dry sieved with a 1 mm mesh sieve. Taxonomic and anatomic identification was carried out using the faunal reference collection available at “BGL Arqueología” lab in Trujillo, Peru, as well as several comparative osteology manuals –51.We used the classical approach of comparative anatomy to estimate the live weight and the length of the fish. The size estimates provided are visual estimates, made by direct comparison with specimens from the fish reference collection of known live weight and length data.In the case of fish, molluscs and other marine invertebrates, species identification can provide information not only on fishing, collecting and hunting practices, but also on changes in the marine and coastal environments around the site.Archaeobotanical studies were performed on the seeds and charcoal collected during the excavations. Each layer of the stratigraphic profile (0–2.80 m) was systematically sampled for archaeobotanical studies, as were two hearths located in layer 35 . The sedTaxonomic identification of the archaeobotanical remains was carried out using the flora reference collection of “BGL Arqueología” lab; F. Moutarde’s collection of wood charcoal from Peru ; A. Chevalier’s Peruvian seed collection ; the International Association of Wood Anatomists’ database InsideWood; as well as several publications, atlases and manuals –59.Preliminary sorting allowed the separation of the different types of botanical remains . The carpological remains were observed using a binocular microscope in order to carry out taxonomic identification based on morphological criteria. Charcoal material from each layer was randomly subsampled at least once, using a riffle box. This first subsampling was done in order to be able to export half of each sample and leave the other half in Peru. The charcoal remains were exported under the permits obtained from the Peruvian Ministry of Culture (No 093-2014-VMPCIC-MC) and studied in the archaeobotanical lab of MSH Mondes, where further subsampling was carried out in order to obtain manageable and comparably sized samples for each layer. Charcoal fragments were studied using a reflective light microscope.The Huaca Grande archaeological sequence sits on a sandy substratum, which was reached at a depth of approximately 2.8 m during the archaeological excavations (Area A4.1). Sediments generally have a sandy texture and are extremely rich in organic debris of various kinds . Layers of mollusc shells and fish remains devoid of matrix alternate with finer, sandy clayey levels that are dark brown in colour and rich in charcoal, burnt seeds and camelid excrement. Whitish powdery layers, composed of ashes, are quite common. Several layers of massive, light grey, sterile sands without any trace of anthropogenic elements have also been identified.Area A4.1 at Huaca Grande gave the most interesting results, test pit A4.2 having only a short sequence with few materials and Cárdenas’s sequence not having been observed in area A4.3 . LocatedA total of 47 layers were identified within the archaeological sequence of profile 4 : (1) at During the excavations of the Huaca Grande mound, we found multiple levels of cultural occupation , which yolla and cántaros) and large (tinajas) jars. At the bottom of the sequence, between layers 43 and 33, we found undecorated and incised ceramics , a necklace of shell beads and a bone pendant, two shell bracelets: one made of spondylus (Spondylus crassisquama) and the another of an undetermined species, and two scallop half valves (Argopecten purpuratus). Ceramics associated with Individual 2 allowed us to determine that this person was affiliated with the Chimú-Inca period  in excavation area A4.3, located in two pits cutting an earthen soil . They wea period .Field observations, in combination with the results of the micromorphological study, made it possible to distinguish 11 sedimentary microfacies (MF) in the Huaca Grande sequence, composed of natural and anthropogenic (occupation) deposits . MFs are(MF1) are the only natural deposits observed at the site. At Nunura Bay, the beach sands are mostly made up of quartz, plagioclases and limestone grains, as well as some rounded shell fragments. MF1 deposits sometimes contain a few reworked anthropogenic elements relating to the site’s occupation, such as fragments of burnt and unburnt bone, shell and charcoal  are mainly composed of burnt and unburnt food waste  and humified organic matter (plant remains and herbivore excrement) mixed with variable amounts of combustion residue  and aeolian sands (MF2 and MF3), but sometimes traces of activity involving the exploitation of specific resources, in particular molluscs (MF4) or fish (MF5), can be recognised  or absence  are living surfaces intentionally constructed with local soils and/or sediments [Earthen floors (or “clay” floors) , based mainly on the mineralogical nature of their silty clayey matrix. Moreover, observations of thin-sections reveal differences in their microstructure and texture, highlighting that different techniques were used for constructing each of these three types of floors .MF9 to MF11) correspond to deposits and structures containing the physical remnants of fire of anthropogenic origin [MF9) and two kinds of reworked residue (MF10 and MF11), the latter two differentiated according to the size of the coarser elements, the size sorting of the burnt residue and the presence or the absence of ash  crystals.Combustion features and waste , horizontal, black (base) and yellow (top) layers bounded by sharp contacts  throughout these layers suggests that they are mostly made up of material resulting from the maintenance of domestic hearths. According to the experiments performed by Miller et al. [r et al. , the coar et al. .In addition to information on the activities carried out on the site, these deposits also reveal the rhythm of its frequentation. All these massive accumulations of burnt materials of types MF10 and MF11 contain very little aeolian sand. They do not show any discontinuity that could indicate a pause in their formation process. These data therefore suggest that (i) the hearths and combustion structures were cleaned regularly and (ii) the site was stably and continuously occupied at the time.When the hearts were not emptied and cleaned, deposits of MF9 type were preserved. Covered by layers of aeolian sands (MF1) or by occupation layers (MF2-type), these deposits attest to the last phase of use of the hearths, either before a period of abandonment or before a new organisation of the site where the combustion structures were located elsewhere or were absent.The 13 radiocarbon dates provide a coherent set, outlining a robust chronological framework for the Huaca Grande archaeological sequence .A Bayesian model was created from these dates, based on the archaeological and stratigraphic data collected during the excavations and on the results of the micromorphological study . ChangesThe resulting model is very robust, as shown by the agreement index , and does not include any outliers. Six main occupation phases and some abandonment periods can be distinguished. Taking into account the median duration calculated from the modelled data , the durIn summary, the chronological data show that the first human settlement started early in the second half of the 5th century CE . The site was then occupied, with one or two brief interruptions, until the second half of the 7th century CE , as attested by the dense succession of combustion structures, occupation layers and accumulations of burnt residue .The deposit of complete camelids and dogs, located between layer 9 and layer 8 (phase 4) and dated between the end of the 9th century and the beginning of the 11th century CE, would have been preceded (after the end of phase 3 –layer 9) and followed (before the beginning of a new domestic occupation–phase 5) by periods of abandonment, each one lasting just over two centuries . These dDomestic human occupation of the site did not recommence until the middle of the 13th century CE, after which it continued until about the middle of the 15th century CE, marking the last human occupation of the site . The modWe analysed 20,446 malacological remains  from Huaca Grande, corresponding to a total weight of 26 kg and representing a minimum number of individuals (MNI) of 12,255 . The levA red square indicates that the taxon lives in the Panamic Province and in the Paita Transition Zone; a blue square indicates that it lives in the Peruvian–Chilean Province. Eco. Bio. = ecological biotope; SM = sandy-muddy; R = rocky; MA = mangrove; Tid. Zone = tidal zone; S = spray zone; H = high-tide zone; M = middle tide zone; Un. = undetermined.Despite the fact that many species of molluscs need specific environmental conditions to thrive, some taxa can occur in more than one biotope , 72. ThuMollusc remains are abundant in the Huaca Grande sequence, but their distribution within the stratigraphic sequence is discontinuous. They occur in layers A, B, 1, 15, 18, 8, 9 , 35, 34, 39, 40 and 41. They are very rare in the oldest layers and become very common in the upper part of the sequence, which corresponds to the most recent occupations. The combined NISP of layers A, B and 1 is 14,846 ; Olivella columellaris, with 3,731 NISP (18.2%); Acanthopleura echinata, with 414 NISP (2%); and Mazatlania fulgurata, with 298 NISP (1.5%)  and an MNI of 7,729.The greatest diversity was found in layer B, where 29 species were identified, and in layers 35, 34 and 39, each yielding 20 species. The most common species in the malacological assemblage are P (1.5%) . The mosScomber japonicus; Scombridae). Croakers (Sciaenidae) show the greatest diversity, being represented by at least five species. Also noteworthy is the good representation of the wrasses (Labridae), which are relatively rare in Peruvian archaeological sites [A preliminary study of ca. 80% of the fish remains that came from area A4.1 permitted us to recognise a rather high diversity of species, mostly characteristic of the modern temperate environment of the Illescas Peninsula. The NISP is 1,332. Sardines (Clupeidae) are the dominant taxon . They aral sites .Merluccius gayi (Merlucciidae), Prionotus stephanophrys (Triglidae) and grunts (Haemulidae). This faunal spectrum seems very close to what could be found along the Illescas Peninsula today.Area A4.1 is characterised by a fairly high proportion of sardines, guitar rays (Rhinobatidae and Batoidea) and sharks (Selachii). This could reflect active fishing with nets, either gillnets or seines, in the sandy bay in front of the site. However, angling was also practised, at least on rocky substrates, as evidenced by the presence of benthic and demersal species, such as moray eels (Muraenidae) and wrasses and the hooks found within the archaeological sequence of the site. Most of the other taxa identified are poorly represented and are essentially demersal taxa: e.g. As far as the sizes of the fish caught are concerned, they are generally small or medium throughout the entire sequence of trench A4.1. Layer 18 is the richest in fish remains and in particular in remains of small pelagic fish, such as sardines and mackerels.The mammal remains from Huaca Grande were separated into two sets: the first comprises the scattered remains of Cetacea, Carnivora, Artiodactyla and Rodentia found in several layers across the stratigraphic sequence, and the second comprises a ritual deposit of camelids and dogs stratigraphically located between layers 9 and 8.Otaria byronia) and South American fur seal  have been identified in the assemblage, the latter being represented by at least four remains. The other taxa are rarer: Camelidae (n = 48), dogs , Cetacea (n = 3) and Rodentia (n = 1).We found 1,601 mammal remains, including 1,325 fragments (82.8% of the NISP) smaller than 5 cm, which corresponds to the total assemblage. They are unevenly distributed along the stratigraphic sequence , occurriThe presence of the remains of 12 camelids and 9 dogs in a pit dug into the top of layer 9 was unexpected. Camelids were found with more frequency in the eastern part of the excavation, while the dogs were recorded in the western part. The specimens were buried either complete or incomplete (without the head), and no specific position or orientation were observed. All the camelids were juveniles, with a biological age estimated from new-born to 9 months. Apart from one adult, all the dogs were juveniles less than 12 months old. No cutmarks were registered neither on camelids and dogs.Phalacrocorax bougainvilliorum, n = 41) and, possibly, neotropic cormorant , plus one Phalacrocorax specimen not attributed to species. The second-most important species is Peruvian booby . Note also the presence of Franklin’s gull , turtledove , Peruvian pelican , and, rarer, Humboldt penguin . Four bird remains were undetermined.Bird remains are uncommon throughout the sequence. We found only 79 remains (NISP), corresponding to a total weight of 700 g . Their dA total of 2,331 pieces of charcoal were recovered and analysed; 1,768 (75.8%) of them were identified to varying taxonomic ranks and assigned to 1 family, 1 subfamily, 1 genus, 1 genus group and 11 species. The number of taxa varies throughout the sequence . Layers Prosopis/Vachellia [Prosopis sp. (american carob) called algarrobo in Spanish, Vachellia macracantha (longspine acacia), known locally as faique and Vachellia aroma (aromatic acacia) var. huarango called espino. They are the most ubiquitous taxa, and they are the only ones present in every layer/feature of the stratigraphic sequence. The second-most ubiquitous taxon is the subfamily of the Caesalpinioideae, also family Fabaceae, present in the lower (layers 41 to 42-L) and middle  parts of the sequence. Parkinsonia sp. and Parkinsonia aculeata  belong to this subfamily and together are the next-most ubiquitous taxa, along with Batis maritima  and Cryptocarpus pyriformis . Other notable taxa, present in one quarter of the samples, are Sesuvium portulacastrum (sea purslane), present in the lower half of the sequence (layers 35-F1 and 34 to 40) and Colicodendron scabridum , present in the upper half of the sequence, particularly in the most recent layers . Other taxa are sporadic, occurring mostly in only a single sample . Caesalpinioideae percentages are much smaller even when we include the sub-taxa (1%–4%). Among all other single taxa, only C. scabridum is appreciably present (3.6%–9%) in layers A, 18 and 8.The Fabaceae also comprise the largest number of specimens, representing 59% to 91.6% of the analysed charcoal in all layers except for the upper ones (B and 1), where they represent only 26% and 7.4% of the samples, respectively. The genus group We analysed a total of 20,065 carpological remains (NISP), i.e. seeds and fruits and fragments thereof. Of these, 1,397 were recovered from dry-sieved samples  and 18,668 from flotation samples (93%). A total of 16,680 remains were identified taxonomically (83%), representing 13 different taxa: 3 genus and 10 species. These percentages show that the two recovery methods led to very different results in the number of collected botanical remains. The flotation method allowed us to retrieve remains smaller than 1 mm and, therefore, to identify a different set of taxa compared with the dry sieving method.Prosopis sp.) occurs in layers 41, 35, 9-M and A, and only one feature, 35-F1, did not yield any carpological remains.Taxonomic diversity is low in most layers, with a tendency of richer layers towards the middle and upper parts of the sequence: layer B presents seven identified taxa, followed by layer 18, with five taxa . Only onProsopis sp.). This taxon is dominant in all the dry-sieved samples, ranging from 99% to 60% of NISP. The second-most ubiquitous taxon is cotton (Gossypium barbadense). It occurs in nine layers, and it is the second-most abundant taxon in terms of NISP, ranging from 1% to 23%. It is unequally present in the lower (layers 34 and 39) and middle (layer 9-M) parts of the sequence, and it is much more frequent, both in ubiquity and in quantity, in the upper part  is present in seven samples, mostly in layer 9  and to a lesser extent in the lower part of the sequence (layers 35 and 40). It has a lower count than Parkinsonia aculeata, which occurs in five layers, in the middle (layers 9-M to 35) and upper (layers B and 18) parts of the sequence. Colicodendron scabridum is also present in five different samples, corresponding to layer 9 (9-U and 9-L) and to the upper part of the sequence.Maize (Cucurbita sp.) and gourd (Lagenaria sp.) are present in two layers each in the top of the sequence . Soapberry (Sapindus saponaria) occurs only in layer 6.Squash . The other two show comparable proportions and are more frequent in layer 9 (9-U and 9-M). Haageocereus versicolor occurs only in layers 9-U and 4 and is particularly frequent in the latter. Oxalis dombeyi is present only in layer 9-M.All other identified taxa were recovered only by flotation . These sOn the basis of field observations and of our multidisciplinary study results, six main phases of human occupation of Huaca Grande can be distinguished ; Fig 13.These architectural and functional features are covered by MF1-type deposits (layer 42), mainly constituted by aeolian sands, including some older anthropogenic elements, such as charcoal fragments, burnt fish bones, mollusc shells or herbivore excrement. These layers hence provide two different types of information. First, they attest that camelids were already present on the site and in the region during this period, as has been noted for the Chicama, Moche and Virú valleys , 79. SecThen, in the following period , up to 1 m thick and consisting exclusively of reworked dumped and swept-out burnt material  indicate that the ritual deposits of animals post-date the formation of the black layer (layer 9). The fill of the trenches where the animals were deposited, as well as the layer (layer 8) that covers them, correspond to sterile aeolian sands (MF1-type), which extend over the entire surface of the excavation area covering the underlying black layer, layer 9. Layer 8 is very irregular in thickness, ranging from a few centimetres along the west profile (profile 4) to more than 50 cm along the east profile (profile 2). This layer testifies to a long period of abandonment of the site, which lasted about six centuries, from the mid-7th century CE to the mid-13th century CE. During this long period, human presence at Huaca Grande is only attested by this deposit of camelids and dogs.Then, a new phase of site occupation is attested by a series of earthen floors  alternating with activity layers , suggesting a continual human presence at Huaca Grande during this phase . The preScomber japonicus gills (In addition to layers indicating generic domestic activities of food preparation and consumption (MF3)–and the presence of small hearths (with a diameter between about 40 and 70 cm) and their maintenance (MF10)–some facies indicating specific activities are observed here. Remains of fish and marine mollusc occur in large amounts in these facies compared with the rest of the archaeological sequence of the Huaca Grande. Fish bones and some shell debris are also present in the lower occupation levels, but they are more scattered there. Here, in contrast, some layers are mainly made up of shell , fish remains , or a mixture of both  and therefore attest to regular catching of fish and gathering of molluscs and their consumption on-site. These activities seem to have replaced mammal hunting as the main component of the subsistence economy. Among these deposits, a concentration of fish remains localised in layer 18 is particularly interesting because it consists exclusively of us gills .This indicates that intensive fishing activities took place and that fish were prepared on-site, probably by removing the heads, viscera and gills, either for immediate consumption or for preservation for later consumption, after smoking or salting. Considering layer 18’s very reduced thickness, the fact that all the remains within it belong to the same species, and its uniqueness within the sequence, its deposition can be considered as an isolated episode. This suggests the absence of a fixed subsistence economy based on a specific resource.Donax obesulus, Acanthopleura echinata and Argopecten purpuratus) in terms of NISP share the same vertical distribution  but not the same habitat (sandy vs rocky). This indicates that they were not the product of a specialised gathering activity. Similarly, there was no systematic exploitation of multiple areas or biotopes; only the nearshore species that were most easily found on the sandy beaches and rocky outcrops near Nunura Bay were collected , suggests that camelids were not used for consumption but that their function was primarily related to trade activities.tinajas), which were probably used for storage. The fill of these pits consists of a sterile grey sandy sediment (MF1), identical to that of layer 8. It therefore seems to attest to a new phase of abandonment of the site, most probably of short duration, as its thickness never exceeds 10 cm. Modelled radiocarbon dates are coherent with this hypothesis, as they suggest that the abandonment lasted about 70 years  and accumulations of shells  and fish remains  alternating with reworked combustion residue . The subsistence economy during this period appears to have been similar to that of the previous occupation phase, based on fishing and shellfish gathering and supplemented with mammals, albeit with a reduced presence of mammals.Cucurbita sp.), or as craft material, such as gourds (Lagenaria sp.). Soapberry fruits also have a varied array of possible uses . None of these plants would have easily grown in numbers in the Nunura Bay area without at least some kind of irrigation and management. It is more likely that they arrived at the site through exchange or trade networks, which seem to have been more intense during the last phases of occupation. It also seems that craft activities using cotton became more important at the time. The most recent date for these occupations ascribes them to the middle of the 15th century CE, and this matches the Chimú-Inca-period date of the two human burials found in area A4.3 . These plants are primarily maize, a food staple, and cotton, central to various crafts, such as the making of cloth or fishing nets. Cucurbitaceae are also of economic importance, either as food, such as squash , are present today in the area surrounding the site. The presence of both temperate-water and tropical species . At this fishing site located at the edge of the Las Salinas palaeolagoon, in the Reventazón area, 35 km southeast of Nunura Bay, warm-water species, such as cephalus , 5. It iSesuvium portulacastrum and Batis maritima are halophytic succulent shrubs commonly found on the coast near the ocean shore, as well as in salt or brackish marshes, salt-pans and mangroves [Tiquilia paronychioides is a common desert shrub in coastal northern Peru, where it grows on sand or sandy clay [quebradas surrounding Huaca Grande.The anthracological and carpological studies show that almost all of the identified taxa are still present in the area surrounding Huaca Grande today. angroves –88, and ndy clay ; it is pProspis sp., B. avicenniifolia, C. scabridum, P. aculeata and S. spicata are xerophytic trees and shrubs characteristic of the dry tropical forest ecological formation of the northern coast of Peru [Prosopis sp. is the more abundant taxon, especially along the quebradas, whereas B. avicennifolia and C. scabridum inhabit the nearby terraces. The rarest is P. aculeata, which is only found in the quebradas. Prosopis/Vachellia is by far the most ubiquitous taxon throughout the archaeological sequence. This can be easily explained by the properties of its wood, being very dense and slow burning, which makes it a very good source of fuel. If available, a combination of Prosopis sp. and V. macracantha usually was the primary firewood used by pre-Hispanic societies on the north Peruvian coast [ of Peru . Among tan coast , 91. TheMaytenus octogona and Cryptocarpus pyriformis are also common xerophytic shrubs of the dry tropical forest. They can be found in areas with a little more humidity, such as desert margins, including the Illescas massif, where they have been found and described (70: 277–278). Nonetheless, neither is present in the vicinity of Nunura Bay nowadays. The presence, although incidental, of C. pyriformis wood in the lower half of the sequence , could reflect less arid conditions around Huaca Grande at the time. However, wood from this shrub could have been gathered farther inland, on the Illescas massif, where it grows today, and brought to the site. Alternatively, it could have been carried down from the Illescas massif by violent floods, like those associated with El Niño episodes; its presence could thus be a marker for this kind of event.Prosopis/Vachellia and Fabaceae proportions decrease significantly only at the end of the occupation sequence (layers A and B). This could be related to (i) a particular activity being carried out using different firewood assemblages; (ii) a change in preservation conditions; or (iii) a decline in the availability of this resource, as a result of either previous overexploitation or a change in environmental conditions. The same hypotheses can be advanced to explain the presence of C. scabridum at the top of the sequence. This shrub is very often found along with Prosopis sp. and is found by itself only in the interior of the desert [C. scabridum over Prosopis sp.The e desert , 93. ThiH. versicolor in layer 4 is consistent with this hypothesis. This Cactaceae is known to be present in the dry tropical forests of the Piura and Lambayeque regions [M. octogona in the same part of the sequence (layer B) would argue against this idea, as it is found in denser and more diverse vegetation. Its presence remains incidental, and it could be the result of a supply from farther away and therefore not reflect local conditions. As explained above, a particular set of activities requiring the use of C. scabridum wood could also explain this occurrence. The presence of Fabaceae and Prosopis/Vachellia in high proportions together with that of C. scabridum in layers 18 and 8 would argue against the preservation hypothesis, according to which the increased proportions of the latter are the result of different preservation conditions.The presence of  regions , 94, altMuntingia calabura grows on disturbed sites in tropical lowlands but is intolerant of saline conditions [Salix humboldtiana grows mainly along river courses and in humid soils. In Piura, it was listed near the main rivers, the Piura and the Chira, and it was also found in the Pomac forest [Only two taxa, found incidentally at the base of the sequence , it is more likely that specimens of these trees came from river valleys farther away, such as the Piura valley. Carried by floods and then deposited on the beach as driftwood, they could have been picked up and used, possibly as firewood. Lastly, it is also possible that wood from these trees was intentionally brought to the area by human groups for specific uses.Further discussion and interpretation of the results on occupation pattern, change in the resources exploited and change in the environments around the site require a comparison with the regional palaeoclimatic record. The occupation of the site corresponds to a period during which the average climate was affected by several variations: i) a wet phase characterised by a high frequency of extreme El Niño events, comparable in intensity to those of 1982/1983 and 1997/1998, which lasted until the middle of the 8th century CE and which is recorded at the base of the sequence, then ii) a phase marked by more arid conditions, which coincides with a weakening of El Niño between 750 and 1250 CE; and, finally, iii) a return to a more humid climate with a resumption of El Niño activity after 1250 CE , 97.Despite several radiocarbon ages that constrain the timing of successive occupation phases at Huaca Grande, the chronological resolution available is not precise enough to establish a clear cause-and-effect link between high-frequency short climatic oscillations (including El Niño events) and periods of human frequentation, successive reorganisations of the site and changes in its function. However, stratigraphical and archaeobotanical data allow us to make some hypotheses.S. portulacastrum remains in layer 9  and especially in layer 35 could result from particular activities involving this plant taking place at that time. However, it could also indicate a larger presence/availability of the plant in the vicinity of the site resulting from environmental changes in Nunura Bay that favoured its growth. One such change could have been the presence or expansion of bodies of salt or brackish water, perhaps related to El Niño floods.Indeed, although the botanical remains attest to a certain stability of the environments around the site over the span of a millennium, multiple clues indicate more occasional, abrupt variations in humidity, probably linked to El Niño-related flooding. In the upper half of the sequence, whose formation occurred from the 7th century CE according to radiocarbon dating, the vast amount of T. paronychioides also significantly increases in the middle and upper parts of layer 9. This could result from wetter and more favourable conditions, possibly due to flooding, than existed during the formation of the lower part of the section and layer 35, where this species is rare. This hypothesis seems to be confirmed by the presence of E. maritimus, which follows the same trend. This Solanaceae is known to rapidly expand in the desert after rain events such as those associated with El Niño and to then die out [The NISP of  die out . We pers die out . This spThroughout the following arid phase, between the middle of the 8th century CE and the middle of the 13th century CE, no permanent occupation took place at the site, and the only traces of human presence relate to the deposition of several animals. This indicates that human groups were still frequenting the bay, but that during this period, it probably did not offer favourable conditions for continuous settlement. Those who deposited the animals were perhaps the human groups (Lambayeque-Sicán culture) living at the nearby site of Huaca Amarilla  or otherpaleteado ceramics and Chimú-Inca type ceramics in the two burials at the top permit to distinguish at least three cultural traditions. The subsistence economies are also different: the earliest group occupying the site used more sea mammals and the latest ones used more molluscs and fish. This pattern is quite different from the groups who settled at Huaca Amarilla, who had access to camelids for their diet, probably due to the occupants of Huaca Amarilla having a higher status. The architecture made of two stone structures, a large domestic area, architecture with coated walls, evidences of storage, burials show that the site of Huaca Amarilla was a civico-ceremonial center of Lambayeque-Sicán culture [Huaca Grande was occupied once again when the climate became wetter, in the middle of the 13th century CE. This settlement phase lasted more or less continuously for two centuries . We can suggest that these populations were linked more strongly with the Huaca Amarilla people and that they seem to be different from the populations of the older occupations. Indeed, the presence of incised ceramics at the bottom of the sequence and of Overall, we can suggest that the phases of wetter average climate and rather intense El Nino activity, up to the middle of the 8th century CE and after 1250 CE, would have allowed for an almost continuous human presence on Nunura Bay, as the local environments would have provided sufficient plant and marine resources to sustain the groups settled on the site. Instead, the arid period between 750 and 1250 CE would have been less favourable for the permanent settlement of human groups in Nunura.The integrated study of the Huaca Grande sequence has permitted us to characterise the occupation of this multifunctional mound over the span of a millennium. Although it has often been identified as a domestic mound, we observed that Huaca Grande did not have a uniform history. It was used successively in several ways, as a domestic, ceremonial, and funerary place. The first human groups who settled on this site, associated with clay public architecture, had an economy turned towards the sea and took advantage of favourable climatic conditions. However, for largely unknown reasons, but partly related to the advent of more arid climatic conditions, the site was abandoned for five centuries. Only the deposition of camelids and dogs interrupted this abandonment. This deposit was a very short-time event and corresponds to some kind of foundation or abandonment offering. In this case, we can suggest that we faced to an event probably linked to the installation of Lambayeque-Sicán nearby site of Huaca Amarilla. Indeed, the chronology of the deposit is coherent with the beginning of Huaca Amarilla occupation around the IX-Xth centuries CE. During the second phase of occupation of Huaca Grande, its inhabitants continue the pattern of exploiting the local natural resources, they acquired other goods (maize and cotton) by exchange, showing that the site had acquired a higher status. The presence of these two taxa is probably linked to the activities that take place in Huaca Amarilla. In this site, 71 burials (70 of immature and one adult) were found and some of which had funerary goods composed by vessels, necklaces and bracelets, and other wood figurine . Some ofS1 Fig(TIF)Click here for additional data file.S1 TableDetailed description of the stratigraphic layers.(XLSX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.


To test this hypothesis, we used computational modeling to quantify the relationships between RyR cluster geometry, phosphorylation patterns, and sarcoplasmic reticulum (SR) Ca2+ release. We found that RyR cluster disruption results in a decrease in spark fidelity and longer sparks with a lower amplitude. Phosphorylation of some RyRs within the cluster can play a compensatory role, recovering healthy spark dynamics. Interestingly, our model predicts that such compensation is critically dependent on the phosphorylation pattern, as phosphorylation localized within the cluster center resulted in longer Ca2+ sparks and higher spark fidelity compared to a uniformly distributed phosphorylation pattern. Our results strongly suggest that both the phosphorylation pattern and nanoscale RyR reorganization are critical determinants of Ca2+ dynamics in HF.Super-resolution imaging techniques have provided a better understanding of the relationship between the nanoscale organization and function of ryanodine receptors (RyRs) in cardiomyocytes. Recent data have indicated that this relationship is disrupted in heart failure (HF), as RyRs are dispersed into smaller and more numerous clusters. However, RyRs are also hyperphosphorylated in this condition, and this is reported to occur preferentially within the cluster centre. Thus, the combined impact of RyR relocalization and sensitization on Ca 2+ during cell excitation. Here, we investigate how the geometry of RyR clusters combined with spatial phosphorylation patterns impacts on Ca2+ spark generation and kinetics. The findings from our study show that phosphorylation pattern and both RyR cluster shape and dispersion have implications on Ca2+ spark activity and provide insights into altered Ca2+ dynamics during HF.Ryanodine receptors (RyRs) are ion channels located on the membrane of the sarcoplasmic reticulum that are responsible for an increase in cytosolic Ca This incoming Ca2+ triggers additional Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) located on the SR membrane  pump, and Ca2+ flux out of the cell through the Na+/Ca2+ exchanger and the plasma membrane Ca2+ -ATPase pump.Excitation-contraction coupling (ECC) refers to a series of electrochemical and mechanical processes that repeat during each heartbeat, and allow coupling between electrical excitation and contraction of the heart . During membrane . This pr2+ homeostasis and contraction, it is essential that this channel’s function is carefully elucidated. It is known that multiple RyRs collaborate to generate Ca2+ sparks, which are the fundamental units of SR Ca2+ release. While the precise number of RyRs that produce a spark has been debated, it is generally accepted that 6–20 channels participate, yielding a release event 10–15 ms in duration  in the cytosolic domain Ωc was:c corresponding to the calcium concentration in the cytosolic domain, Dc the diffusion constant of calcium, bi the concentration of the corresponding buffer and s , one calsequestrin buffer was included and its concentration will be denoted by b5. The following equations correspond to the spatio-temporal evolution of [Ca2+] in the SR domain Ωs:s corresponding to the calcium concentration in the SR domain. Both domains were coupled at the SR membrane with the following boundary conditions:The mathematical model from the work of Kolstad et al.  was adapet al. , the mod [et al.  and incl [et al.  for bothet al. [JRyR and JpRyR formulations were identical to the JRyR flux formulation from Kolstad et al. [The SERCA flux formulation was taken from the three-state SERCA model from Tran et al. . The JRyd et al. , where ak− and k+ variables were defined as:Here, C describes the conductive state and O the non-conductive state. The JRyR and JpRyR is the value used for K+. The choice of the parameter K+ for the phosphorylated and the non phosphorylated case is discussed in Section “K+ parameter estimation”. The difference between c ∩ Ωs). We assumed that the simulated CRU was a 3D cube with volume 1.008 μm × 1.008 μm × 1.008 μm. Cube dimensions were 12 nm × 12 nm × 12 nm (Δx = 12 nm) with 84 voxels per axis.The model consists of a single CRU containing both cytosolic and SR domains  around the RyRs as described in .2+ spark signals, three different phosphorylation patterns were applied , we sim2+ [K+ value for a phosphorylated receptor . For the no phosphorylation condition, see red dots in K+ value was set to 55 μm. This value was chosen by optimizing K+ values to achieve physiologically realistic spark property values [K+ value was set to 25 μm. This K+ value was estimated based on obtaining sparks with spark properties within the same range as previous experimental results [K+ values for blanket phosphorylation was set such that the Ca2+ dynamics from a blanket phosphorylation pattern would match the Ca2+ kinetics obtained from using a uniform pattern. Computational studies generally assume all RyRs in a CRU to be identically sensitized [K+ values for the blanket phosphorylation case, see K+ was set to 45 to compare a blanket phosphorylation with a 20% uniform phosphorylation. Additionally, K+ was set to 35 to compare a blanket phosphorylation with a 50% uniform phosphorylation. An overview of the chosen K+ values is given in K+ values were kept constant through all 5 different geometries, such that the only difference between the different geometries is the spatial localization of the RyRs.RyR phosphorylation increases the channel sensitivity to Ca2+ ; we simuy values . A partiy values . For pho results , 21. Thensitized –16. Thernsitized . Since w2+ concentrations. The simulations were terminated when all RyRs were closed and remained closed for 1 ms. Therefore, we define spark duration as the duration of the simulations 1 ms after all RyRs are closed. Since the model is computationally intensive, simulations were generally terminated 1 ms after all RyRs, both phosphorylated and non phosphorylated, were closed, a point at which relevant metrics could be attained. After all RyRs are closed, the remainder of the spark is modeled as an exponential decay function which is independent of the RyR cluster spatial organization and the phosphorylation pattern. In 2+ spark. The stochastic RyR model was calculated for a fixed time step of Δt = 1 ms.Since the model used assumes a stochastic model for RyR opening, 200 runs were carried out for each of the 40 configuration setups (5 geometries × 8 phosphorylation patterns). The simulations were initiated by randomly opening 1 RyR within the specific geometry, and the RyR fluxes were then calculated. Opening and closing of a single RyR was based on local Ca2+ fluorescence intensity exceeded 30% (ΔF/F0 ≥ 0.3), and used the fraction of these successful sparks to estimate spark fidelity. The confidence interval of spark fidelity was calculated using the Agresti-Coull confidence interval [The model calculations were very stiff due to the small element volumes and the large fluxes. Therefore, the calculations of the fluxes in the model, described in the section “Model development”, were solved analytically in the same fashion as . We alsointerval . Additioet al. [0 > 0.3) is observed between these two geometries  as opposed to some percentage of uniformly phosphorylated receptors in the cluster . The darker shades represent a 50% uniform phosphorylation pattern (dark red) and blanket phosphorylation with a K+ value of 35 (dark gray). We find that, by adjusting K+ values, outputs from blanket phosphorylation match those from uniform phosphorylation for all analyzed spark properties , despite the shorter TTP see . Importaet al. [We next analyzed the effects of RyR phosphorylation in disrupted CRU geometries, which are reported in HF . As noteet al. , during We first focus on the differences between G2 and G3, since the only difference between these geometries is that G2 contains one cluster and G3 two sub-clusters, while overall cluster shape is maintained . For botHowever, differing effects on spark amplitude were observed for compact geometry G2 and discontinuous geometry G3 . Indeed,2+ between subclusters was much less frequent with an outer phosphorylation pattern, with a lower number of RyRs being activated  [et al. [et al. [et al., healthy compact geometries are compared with phosphorylated disrupted geometries. In our study we also saw that disrupted geometries undergoing phosphorylation could lead to longer and more frequent sparks than compact geometries without phosphorylation  . Super ral RyRs) . In a re [et al. , they ob [et al. , where drylation . For exa  may hin2+ spark properties. Additionally, these results show the large impact that spatial phosphorylation pattern can have on Ca2+ spark properties, leading to differences in spark fidelity, amplitude or duration depending on the pattern. It is necessary to perform further studies, both computational and experimental, to explore larger-scale impacts of RyR phosphorylation patterns on whole cell calcium dynamics. For example, a computational study including multiple nearby clusters may address whether specific phosphorylation patterns may allow for more reliable spread of calcium sparks into calcium waves through the dyadic region. Additionally, further simulations including an increase in the volume of the dyadic junction due to t-tubule loss in heart failure would be interesting. We believe that an increased volume should lead to a desensitization of the RyR channels to calcium and the effect observed during RyR disruption should be even more pronounced. Further imaging studies based on the spatial distribution of the phosphorylated RyRs as shown in [2+ sparks [In this study we reaffirm the importance of cluster geometry on Cashown in  and on tshown in , 29 shou+ sparks , but alsS1 FigThe arrows are vectors showing the eigenvectors of the covariance matrix of the spatial distribution of the RyRs. A first ellipse around the eigenvectors is shown. The ellipse dimensions are decreased  until the desired number of phosphorylated RyRs is reached (in this case 50%).(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(TIFF)Click here for additional data file.S5 FigFor each geometry 200 simulations were conducted. (A) The distribution created by the relation between TTP and amplitude is shown in a scatter plot shows. The crosses represent the mean values across all simulations. (B) The spark fidelity for each geometry is presented in a bar chart.(TIFF)Click here for additional data file.S6 FigThe red circle indicates which RyR was opened to start the simulation.(TIFF)Click here for additional data file.S7 FigPlease note that the bars show relative values; The parameter values for the non phosphorylated case were normalized to 100% and the values shown here represent the relative increase with respect to the non phosphorylated case.(TIFF)Click here for additional data file.S8 FigOn the left, the histogram for G1 geometry is shown, on the right the histogram for G2 geometry.(TIFF)Click here for additional data file.S1 Table(TIFF)Click here for additional data file.S2 Table(TIFF)Click here for additional data file.S3 Table(TIFF)Click here for additional data file.S1 Video(MP4)Click here for additional data file.S2 Video(MP4)Click here for additional data file.


Saccharomyces cerevisiae by producing its precursor diacylglycerol and concurrently regulates de novo phospholipid synthesis by consuming its precursor PA. The function of Pah1 requires its membrane localization, which is controlled by its phosphorylation state. Pah1 is dephosphorylated by the Nem1-Spo7 protein phosphatase, whereas its phosphorylation occurs by multiple known and unknown protein kinases. In this work, we show that Rim11, a yeast homolog of mammalian glycogen synthase kinase-3β, is a protein kinase that phosphorylates Pah1 on serine  and threonine  residues. Enzymological characterization of Rim11 showed that its Km for Pah1 (0.4 μM) is similar to those of other Pah1-phosphorylating protein kinases, but its Km for ATP (30 μM) is significantly higher than those of these same kinases. Furthermore, we demonstrate Rim11 phosphorylation of Pah1 does not require substrate prephosphorylation but was increased ∼2-fold upon its prephosphorylation by the Pho85-Pho80 protein kinase. In addition, we show Rim11-phosphorylated Pah1 was a substrate for dephosphorylation by Nem1-Spo7. Finally, we demonstrate the Rim11 phosphorylation of Pah1 exerted an inhibitory effect on its PA phosphatase activity by reduction of its catalytic efficiency. Mutational analysis of the major phosphorylation sites  indicated that Rim11-mediated phosphorylation at these sites was required to ensure Nem1-Spo7-dependent localization of the enzyme to the membrane. Overall, these findings advance our understanding of the phosphorylation-mediated regulation of Pah1 function in lipid synthesis.Pah1 phosphatidate (PA) phosphatase plays a major role in triacylglycerol synthesis in  Saccharomyces cerevisiae, Pah1 is a Mg2+-dependent phosphatidate (PA) phosphatase (PAP) catalyzing the conversion of PA to diacylglycerol (DAG) (de novo synthesis of membrane phospholipids (In the yeast ol (DAG)   regulatory circuit  motif are essential for catalytic activity  lie in the same plane (B), suggesting the importance of their alignment on the membrane surface for the enzyme to recognize its substrate (B).The two conserved domains  of Pah1 , 28 are activity , 28, 60 activity . In addiactivity  located activity , 61. Trpactivity , 61. Accactivity , Trp637 me plane B, suggesubstrate B.Figure Pah1 is easily degraded by the 20S proteasome when it is dephosphorylated (or unphosphorylated) but stable against the proteasomal degradation when it is phosphorylated , 64. An A). Many of those sites have been identified as target residues for protein kinases such as Cdc28-cyclin B . The protein kinase–target site relationship is important to understand the role of a specific phosphorylation in the regulation of Pah1 for its location, PAP activity, and protein stability , its phosphorylated form  showed a decrease in electrophoretic mobility. To better determine the mobility shift, Pah1 was resolved in the gel containing Phos-tag, a molecule that traps phosphorylated proteins. In the Phos-tag gel, unphosphorylated Pah1 migrated as a single discrete band , whereas the protein phosphorylated by Rim11 showed multiple bands of slower migration , indicating heterogeneous phosphorylation of the protein. Compared with the Rim11-phosphorylated Pah1, the protein phosphorylated endogenously in yeast showed more bands of slower electrophoretic mobility , indicating that it is highly and heterogeneously phosphorylated in the cell.To examine Pah1 phosphorylation by Rim11, we utilized the protein purified from its heterologous expression in in yeast , 61. We 32P]ATP into the protein. In the assay, the 32P-labeled Pah1 was resolved from [γ-32P]ATP by SDS-PAGE and then quantified by phosphorimaging (B). Phosphopeptide mapping of the 32P-labeled Pah1 indicated that its phosphorylation by Rim11 occurs on multiple sites (C), and phosphoamino acid analysis showed that its phosphorylation occurs on the serine and threonine residues with the former being a major target site .Although the electrophoretic mobility is useful to assess the phosphorylation state of Pah1, it is not as sensitive and quantitative as enzyme assays  that mearimaging B. Phosphle sites C, and phget site D, right.D, right).The phosphorylation sites of Pah1 were identified by LC-MS/MS analysis of phosphopeptides derived from its digestion with trypsin, chymotrypsin, and Glu-C . Based oFig. 4A and B). In addition, the Rim11 activity was dependent on the concentrations of Pah1 and ATP (C and D). The Vmax and Km values of Rim11 for Pah1 were 169 units/mg and 0.4 μM, respectively, and those for ATP were 253 units/mg and 30 μM, respectively. At the point of maximum phosphorylation, Rim11 catalyzed the incorporation of 2 mol of phosphate/mol of Pah1. Overall, these enzymological properties demonstrate that Pah1 is a bona fide substrate of Rim11.The Rim11 protein kinase was further characterized with Pah1 as substrate. The enzyme activity was dependent on reaction time and the amount of Rim11, indicating that the protein kinase follows zero order kinetics , A and B and ATP , C and DE. coli-expressed Pah1 was phosphorylated by Rim11 and then measured for its PAP activity with respect to the surface concentration of PA in Triton X-100/PA-mixed micelles (n = 3.4) with respect to PA  and PAP activity with the inhibitory effect stronger at the higher concentrations of PA. The Vmax value (1.2 μmol/min/mg) of the phosphorylated Pah1 was 1.8-fold lower than that of its unphosphorylated form. However, the Km value (4.5 mol %) of the phosphorylated enzyme for the substrate PA was not significantly different from that of the unphosphorylated form. These kinetic data indicate that the Rim11-mediated inhibition of Pah1 PAP occurs by decreasing its catalytic efficiency.We examined the PAP activity of Pah1 phosphorylated by Rim11. The micelles , 76. Undmicelles , the PAPct to PA . The VmaXXXS/T by another protein kinase  Pah1 was phosphorylated first by Pho85-Pho80 with nonradioactive ATP and then by Rim11 with [γ-32P]ATP. The measurement of radioactive Pah1 showed that its phosphorylation by Pho85-Pho80 stimulated (∼2-fold) the subsequent phosphorylation by Rim11 XXXS/T byby Rim11 . In the by Rim11 . In thisby Rim11 , 75, wasby Rim11 ; phosphoE. coli-expressed Pah1 was phosphorylated by Rim11 with [γ-32P]ATP to synthesize a radiolabeled substrate. The 32P-labeled Pah1 was mixed with the Nem1-Spo7 complex reconstituted in phospholipid vesicles, and the phosphatase activity was scored by the release of 32Pi from the radioactive protein  of Pah1 phosphorylation by Rim11 and examined the physiological consequences of the phosphorylation deficiency. The phosphorylation-deficient alleles of PAH1  were expressed on a low-copy plasmid in the pah1Δ and pah1Δ nem1Δ strains. The pah1Δ nem1Δ mutant, which lacks the Nem1 protein phosphatase catalytic subunit . In addition, we examined whether the localization of Pah1 is affected by the lack of its phosphorylation by Rim11. As described previously . The TAG content was not significantly affected by the Rim11 phosphorylation-deficient mutations. When wildtype Pah1 was expressed in pah1Δ nem1Δ cells, TAG accounted for only 2% of the total lipids (B). This result confirms previous work showing that the dephosphorylation of Pah1 by the Nem1-Spo7 protein phosphatase complex is crucial for its function in TAG synthesis (pah1Δ nem1Δ cells expressing the Rim11 phosphorylation-deficient mutant forms of Pah1 was 1.4- to 2-fold higher when compared with that of the cells expressing the wildtype Pah1 (B). This result indicates that the lack of Pah1 phosphorylation on Thr163, Thr164, and Ser602 partially bypasses the requirement of the Nem1-Spo7 complex for its localization to the membrane.As the effect of Pah1 PAP activity on TAG content is pronounced in stationary phase cells , 11, 17,d lipids A. The TAl lipids B. This rynthesis , 80. Theype Pah1 B. This rRIM11 were grown in SC medium from the exponential to the stationary phase, and the cell extracts were analyzed for Rim11 abundance by Western blotting with anti-protein A antibody . Considering that one protein kinase is not sufficient to properly phosphorylate Pah1, the employment of multiple protein kinases provides a mechanism to ensure its phosphorylation for the Nem1-Spo7-dependent regulation. In this regard, Ser602 is shown to be a site whose phosphorylation is secured by Rim11, casein kinase I Km value XXXS/T XXXS/T  are involved in the control of Pah1 function is a question that needs to be addressed to better understand its regulation by phosphorylation.Rim11 phosphorylates Pah1 mainly on Ser602, which is one of the seven sites required for its membrane translocation by Nem1-Spo7 protein phosphatase complex. Accordingly, Pah1 deficient in the phosphorylation site bypasses the requirement of Nem1-Spo7 as indicated by its partial functional role in the S. cerevisiae strain BY4741-RIM11-TAP were purchased from Thermo Fisher Scientific. Phos-tag Acrylamide AAL-107 was purchased from Wako Chemicals.Chemicals were reagent grade. Lipids and silica gel GHL TLC plates, respectively, were from Avanti Polar Lipids and Analtech. Bio-Rad was the supplier of DNA size ladders, molecular mass protein standards, and reagents for electrophoresis and Western blotting. Cayman Chemical was the source of leupeptin and pepstatin. Clontech supplied carrier DNA for yeast transformations. Growth media were obtained from Difco Laboratories. Expedeon was the source of InstantBlue (Coomassie blue) protein stain. GE Healthcare was the source of Q-Sepharose, IgG-Sepharose, PVDF membrane, and the chemifluorescence Western blotting detection kit. Invitrogen supplied mouse anti-phosphoglycerate kinase antibodies . Millipore Sigma was the source of ATP, bovine serum albumin, cellulose TLC plates, chymotrypsin, phosphoamino acid standards, protease and phosphatase inhibitors, Ponceau S stain, L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin, Triton X-100, rabbit anti-calmodulin binding protein epitope tag antibody , rabbit anti-protein A antibody , and alkaline phosphatase–conjugated goat anti-mouse IgG antibodies . Scintillation counting supplies were from National Diagnostics. Q5 site-directed mutagenesis kit and other reagents for DNA manipulations were from New England Biolabs. Radiochemicals were purchased from PerkinElmer Life Sciences. Qiagen supplied DNA gel extraction and plasmid purification kits and the nickel-nitrilotriacetic acid agarose resin. Pierce mass spectrometry grade proteases and strong anion exchange spin columns, alkaline phosphatase–conjugated goat anti-rabbit IgG antibody , and S. cerevisiae strain W303-1A was the source of the RIM11 gene and used for the construction of the rim11Δ mutant SKY001 and for the expression of plasmid-borne RIM11. BY4741-RIM11-TAP was the source of the RIM11-TAP gene that was cloned into pYES2, a galactose-inducible overexpression plasmid, for the construction of pSK001. E. coli strains DH5α and BL21(DE3)pLysS were used for the propagation of plasmids and heterologous expression of His6-tagged Pah1, respectively.The yeast and bacterial strains used in this study are listed in PAH1 (T163A/T164A), PAH1 (S602A), and PAH1 (T163A/T164A/S602A) were generated from pGH315 using the Q5 Site-directed mutagenesis kit. The RIM11 gene was amplified by PCR with the addition of NotI/XhoI sites from the W303-1A genomic DNA and inserted into the NotI/XhoI sites of plasmid pRS415 to construct pSK003. Plasmid pSK001 was generated by inserting the RIM11-TAP sequence, which was PCR amplified from BY4741-RIM11-TAP DNA, into pYES2 at the KpnI/NotI sites. The rim11Δ mutant strain SKY001 was constructed from W303-1A by homologous recombination of the rim11Δ::KanMX cassette that was PCR amplified from the yeast mutant collection. Standard methods were used for isolation of chromosomal and plasmid DNA, for digestion and ligation of DNA, and for PCR amplification of DNA  using a spectrophotometer. Yeast cells carrying a plasmid were grown at 30 °C in synthetic drop-out medium, which lacks a specific amino acid from synthetic complete (SC) medium for the plasmid selection. Cells of strain W303-1A harboring plasmid pSK001 were inoculated into 250 ml of SC-Ura/2% glucose medium to a final cell density of A600 ∼ 0.1 and then grown to saturation. The saturated culture was harvested by centrifugation at 1500g for 10 min and the cell pellet was resuspended (A600 ∼ 0.4) in 2 L of SC-Ura/1% raffinose/2% galactose medium and incubated for 14 h (A600 ∼ 1.0) with shaking at 250 rpm. The E. coli cells were grown at 37 °C in lysogeny broth (LB) medium ; ampicillin (100 μg/ml) was added to select for cells carrying plasmids. For His6-tagged Pah1 expression, the bacterial cells harboring plasmid pGH313 were grown to A600 ∼ 0.5 at room temperature in 500 ml of LB medium containing ampicillin (100 μg/ml) and chloramphenicol (34 μg/ml); expression was induced for 1 h with 1 mM isopropyl-β-D-thiogalactoside  buffer containing 0.3 M sucrose, 10 mM 2-mercaptoethanol, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 5 μg/ml pepstatin. Glass beads (0.5 mm diameter) were added to cell suspensions and then subjected to five repeats of 1-min burst and 2-min cooling using a BioSpec Products Mini-Beadbeater-16 (g for 10 min to separate unbroken cells and cell debris (pellet) from cell extracts (supernatant). The cell extract was centrifuged at 100,000g for 1 h to separate the cytosol (supernatant) from the membrane (pellet). The membrane fraction was resuspended in the same volume of cell disruption buffer.All steps were performed at 4 °C. Yeast cultures were harvested by centrifugation at 1500eater-16 . The dis6-tagged Pah1 expressed in E. coli was purified from cell extracts by affinity chromatography with nickel-nitrilotriacetic acid-agarose  buffer containing 150 mM NaCl, 1 mM EDTA, Roche EDTA-free protease inhibitors, and 2% Triton X-100 and centrifuged at 100,000g for 1 h. The supernatant was applied to a 0.5-ml IgG-Sepharose column equilibrated with 50 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl, 0.5 mM EDTA, and 0.1% Triton X-100. The column was washed with 20 column volumes of the equilibration buffer and then incubated at room temperature for 1 h with 75 units of tobacco etch virus protease in 0.5 ml 50 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl, 0.5 mM EDTA, 0.1% Triton X-100, 1 mM dithiothreitol. The protein A–free Rim11 was eluted from the IgG-Sepharose resin with the chromatography buffer. The tobacco etch virus protease was removed from the Rim11 preparation by passage through a strong anion exchange spin column equilibrated with 20 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl and 10% glycerol. Rim11 was then eluted from the spin column by increasing the NaCl concentration in the equilibration buffer to 250 mM. Purified Rim11 preparations were stored at −80 °C.All steps were performed at 4 °C unless otherwise indicated. The galactose-induced yeast culture of W303-1A expressing the TAP-tagged Rim11 was harvested, and the cell pellet was resuspended in 16 ml of 50 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl, 1 mM EDTA, and Roche EDTA-free protease inhibitors and lysed with glass beads using a Mini-Beadbeater-16 (5 repeats of 1-min burst with 2-min cooling between bursts). The cell lysate was centrifuged at 15006-tagged Pho85-Pho80 complex was purified from E. coli BL21(DE3) cells that expressed plasmids EB1164 and EB1076 by affinity chromatography with nickel-nitrilotriacetic acid-agarose , 0.25 μM Pah1, 2 mM dithiothreitol, and the indicated amount of Rim11. The phosphorylation reaction was terminated by the addition of 6.7 μl 4x Laemmli sample buffer, followed by SDS-PAGE , and the extent of phosphorylation was quantified by ImageQuant software.Rim11 protein kinase activity was measured at 30 °C by following the incorporation of radioactive phosphate from ATP as described by Carman and Lin  and [γ-32P]ATP  for 2 h at 30 °C with Rim11 (300 ng) or Pho85-Pho80 (1.7 μg). The protein kinase and nucleotide were removed from the phosphorylated Pah1 by use of a strong anion exchange spin column  with the accession number MSV000088038. All other data are contained within the article or the supporting information.Raw MS phosphorylation data and database search results for Pah1, along with the Rim11 analysis data are deposited in the MassIVE repository (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Atopic dermatitis (AD) is a chronic pruritic skin disease with a complex pathogenesis underlying its heterogeneous clinical phenotypes and endotypes. The skin manifestation of AD reflects the cytokine milieu of a type-2-dominant immunity axis induced by genetic predisposition, innate immunity dysregulation, epidermal barrier defects, and allergic inflammation. However, the detailed pathomechanism of eczematous dermatitis, which is the principal characteristic of AD, remains unclear. This review examines previous studies demonstrating research progress in this area and considers the immunological pathomechanism of “spongiotic dermatitis”, which is the histopathological hallmark of eczematous dermatitis. Studies in this field have revealed the importance of IgE-mediated delayed-type hypersensitivity, the Fas/Fas-ligand system, and cell-mediated cytotoxicity in inducing the apoptosis of keratinocytes in spongiotic dermatitis. Recent studies have demonstrated that, together with infiltrating CD4 T cells, IgE-expressing dendritic cells  that capture specific allergens  are present in the spongiotic epidermis of lichenified eczema in patients with IgE-allergic AD. These findings suggest that IgE-mediated delayed-type hypersensitivity plays a pivotal role in the pathogenesis of spongiotic dermatitis in the skin lesions of AD. Staphylococcus aureus (S. aureus) ,862 in aBy definition, acute lesions of AD are characterized by severe pruritic papules on extensively erythematous skin, accompanied by serous exudates . HoweverIn APT-positive reaction sites for HDM antigens in the non-lesioned skin of IgE-allergic AD patients c, HDM anRegarding the relationship between spongiosis and the localization of HDM antigens in APT-positive reaction sites, we reported a case in which HDM antigens were not captured by IgE+ IDECs in the central area of a spongiotic vesicle, but instead by IgE+ LCs in the area around the spongiotic vesicle f 86]. H. H86]. H2 in a 200× visual field)  i 76]. T. T2 in al field) j, and IgInterestingly, we encountered a case of IgE-allergic AD with lichenified eczema, in which co-expression of HDM antigens was observed only with IgE+ LCs in the areas around spongiotic vesicles and not with IgE+ IDECs in the central area of the lesion k,l, simiIFITMI gene expression is significantly higher in keratinocytes of AD patients [By using schematics a,b, thispatients . These cpatients . Howeverpatients ,50. Assopatients  due to tpatients ; transaipatients ; or tranpatients ,47,60. IIn lesioned skin b, lichenn = 7, range; 1.8 to 13.7%) of T cells infiltrating into the spongiotic epidermis express IL-13 [In general, infiltration of Th1 cells requires IL-12 secretion within lesion tissues, and macrophages, eosinophils, and DCs  are known sources of this cytokine ,109. Howss IL-13 . INF-γ sss IL-13 . Hence, Antigens in allergic skin diseases such as ACD are generally presented by MHC-class II (human leukocyte antigen–DR) on keratinocytes as haptens with carrier proteins, and lymphocytes react with these antigens to form spongiosis lesions . TherefoIn general, AD is roughly divided into two distinct variants depending on the presence or absence of involvement in the pathology of IgE allergy: (1) extrinsic IgE-allergic AD, which occurs in the context of sensitization to environmental allergens that produce allergen-specific IgEs and is accompanied by elevated serum total IgE levels , and (2) intrinsic non–IgE-allergic AD, with no detectable sensitization to environmental allergens and with normal serum total IgE levels . In the S. aureus, as well as environmental allergens, such as HDMs, pollen, and fungi [The concept of spongiotic dermatitis associated with IgE-mediated delayed-type hypersensitivity described above is based upon the presence of IgE+ LCs and IgE+ IDECs ,56. Prevnd fungi ,114,115.nd fungi ,68. Thesnd fungi .With regard to the characteristics of patients with non–IgE-allergic AD, the following cytokine profiles of serum and skin lesions have been reported. In comparison to patients with IgE-allergic AD, serum IL-13 levels are higher in patients with non–IgE-allergic AD, whereas serum IL-5 levels are higher in patients with IgE-allergic AD, and the serum IL-4 and soluble IL-4Rα levels are the same in these patients . In contIt is expected that clinical practice and medical treatment of AD will be carried out in line with the understanding and stratification of endotypes and phenotypes based on molecular taxonomy data ,118,119.