Mast cell mediators released during the allergy effector phase drive allergic reactions. Therefore, targeting sensitized mast cells may prevent food allergy symptoms.in vitro system to evaluate potential therapeutics targeting sensitized effector cells.We used novel, human, allergen-specific, IgE monoclonal antibodies (mAbs) created using human hybridoma techniques to design an Two human IgE mAbs specific for peanut, generated through human hybridoma techniques, were used to sensitize rat basophilic leukemia (RBL) SX-38 cells expressing the human IgE receptor (FcϵRI). Beta-hexosaminidase release (a marker of degranulation), cytokine production, and phosphorylation of signal transduction proteins downstream of FcϵRI were measured after stimulation with peanut. Degranulation was also measured after engaging inhibitory receptors CD300a and Siglec-8.Peanut-specific human IgE mAbs bound FcϵRI, triggering degranulation after stimulation with peanut in RBL SX-38 cells. Sensitized RBL SX-38 cells stimulated with peanut increased levels of phosphorylated SYK and ERK, signal transduction proteins downstream of FcϵRI. Engaging inhibitory cell surface receptors CD300a or Siglec-8 blunted peanut-specific activation.in vitro models of the effector phase of food allergy. Peanut reproducibly activates and induces degranulation of RBL SX-38 cells sensitized with peanut-specific human IgE mAbs. This system provides a unique screening tool to assess the efficacy of therapeutics that target allergy effector cells and inhibit food allergen-induced effector cell activation.Allergen-specific human IgE mAbs, expressed from human hybridomas and specific for a clinically relevant food allergen, passively sensitize allergy effector cells central to the  Food allergies affect 10% of the US population, including 2%, or over 6 million people, with peanut allergies .de novo lipid mediators and additional cytokines to maintain allergic symptoms  during the allergy effector phase . During symptoms . Thus, tin vitro and suppress anaphylaxis in siglec-8 transgenic murine models -8 mitigate allergic inflammation and MC degranulation in passive cutaneous anaphylaxis models , 6 and ain vitro assays that consistently demonstrate the ability of a clinically relevant food allergen to induce MC degranulation. Current in vitro models of the allergy effector phase use purified human IgE antibodies whose antigen specificities are unknown  created via human hybridoma techniques , dust mite, and Der p 2 Table E1+ RBL SX-38 cells was 93.2% when cells were sensitized simultaneously with both peanut IgE mAbs   SX-38 cells , commonlyssinus)  and examTo evaluate the ability of peanut IgE mAbs to induce allergen-specific effector cell degranulation, we sensitized RBL SX-38 cells overnight with peanut or Der p 2-specific IgE mAbs. Cells were stimulated with either whole peanut extract or Der p 2 antigen at varying concentrations and beta-hexosaminidase release was measured. RBL SX-38 cells sensitized with peanut IgE mAbs degranulated in a dose-dependent manner following stimulation with peanut extract  (Il4 (Il6 (Cox2 (Mcp1 (p=0.0526) and Tnfa (p=0.0681) gene expression in response to Ara h 2 stimulation .Four-hour stimulation with peanut led to statistically significant increases in Mcp1 . However, increased amounts of GM-CSF were produced by cells sensitized with peanut IgE mAbs and stimulated with 1 µg/mL peanut or 1 µg/mL Ara h 2 antigen compared to unstimulated cells -containing inhibitory mast cell surface receptors have been proposed as possible pharmaceutical targets to block mast cell activation and degranulation in food allergy . We obsein vitro , 31, 32 o models .ITIM-containing inhibitory receptors act through ITIMs by recruiting phosphatases to dephosphorylate signaling proteins downstream of the FcϵRI . The mecin vitro and in vivo systems for subsequent evaluation of candidate inhibitors, since the influence of polyclonal IgE, other antibody isotypes, and non-immunoglobulin components like complement should be taken into account when developing a mast cell inhibitor applicable to allergic human patients. Our method deploys novel peanut-specific human IgE monoclonal antibodies to enable reproducible in vitro models of the effector phase in food allergy as a first step in the effort to unveil new therapeutic targets to prevent mast cell activation in IgE-mediated allergy. Additional studies are required to determine the clinical relevance and the mechanisms driving the inhibition observed; however, our findings represent a key starting point for identifying potential therapeutic targets on MCs and basophils and understanding how they may blunt allergen-induced effector cell activation.Our system allows for observation of the direct effects of potential therapeutics on effector cell activation by removing variables introduced when human plasma is used for sensitization. Because it excludes non-relevant IgE, multiclonal allergen-specific IgE, and other antibody subclasses, our platform simplifies the allergic effector phase in food allergy and facilitates the rapid screening of candidates for effector cell inhibition in food allergy. By no means does our model obviate the role for more complex 2. Cells were cultured in 1X MEM , supplemented with 1.2 mg/mL geneticin (Gibco-Thermofisher), 2mM L-glutamine, 100 U/mL penicillin and 100 ug/mL streptomycin (Gibco-Thermofisher), and 10% heat-inactivated Cytiva Hyclone FBS . During overnight sensitization steps for all experiments, complete media was replaced with media without geneticin and Cytiva FBS as previously described  and the cells were shared with us by Dr. Michelle Hernandez (Chapel Hill). RBL SX-38 cells were maintained at 37° C in 5% COescribed .Peanut IgE mAbs  and Der p 2 IgE mAbs (2F10 and 4C8) were developed and gifted by Scott A. Smith using human hybridoma techniques as previously described  and are 6 RBL SX-38 cells/condition were sensitized overnight with peanut IgE mAbs (500 ng/mL each) or Der p 2 IgE mAbs (500 ng/mL each) in the presence or absence of 250 nM omalizumab . Cells were then washed and stained with eBioscience Fixable Viability Dye eFluor 450 (Thermofisher) and human IgE clone MHE-18-Alexa Fluor 647 . Cells were washed again, fixed, and acquired on an Attune NxT acoustic focusing cytometer (Life Technologies-Thermofisher). Flow cytometry data were analyzed using FlowJo software .Approximately 1 x 10Peanut Extract- Peanut extract was prepared by extracting peanut protein from roasted defatted peanut flour  as previously described  allergen was purchased from Indoor Biotechnologies .6 RBL SX-38 cells were sensitized with two peanut IgE mAb clones  or two Der p 2 IgE mAb clones . Cells were then washed with NaPIPES buffer  before stimulating with 1 µg/mL whole peanut extract, Der p 2, or buffer for 5 minutes. Stimulated cells were lysed on ice for 30 minutes. A bicinchonic acid (BCA) protein assay was performed on cell lysates to determine total protein content. Equal amounts of total protein were loaded onto SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in tris buffered saline with tween (TBST) with 2% bovine serum albumin (BSA) for 1 hour before incubating overnight with primary antibodies: 1:1000 pSYK Antibody #2711 , 1:1000 pERK1/ERK2 , 1:1000 total SYK , or 1:500 total ERK1/2 . Membranes were washed with TBST, then incubated for 1 hour with secondary antibodies: 1:1000 Goat Anti-Rabbit IgG  or 1:1000 Goat Anti-Mouse IgG1-HRP . Chemiluminescence was used for detection of blots via a Bio-Rad Chemidoc .2 x 106 cells/mL). Cells were incubated overnight with 1 µg/mL Peanut IgE mAbs  or Der p 2 IgE mAbs , followed by a 45-minute stimulation with different stimuli. Supernatants and cell lysates were then incubated with a substrate for 1 hour, followed by 0.2 M glycine buffer immediately before colorimetric detection. Optical density (OD) values were determined at 450 nm and percentage beta-hex release was calculated as a measure of degranulation. For inhibition studies, sensitized cells were stimulated and immediately treated with 0.1, 1, or 10 µg/mL Rabbit IgG isotype control , 0.1, 1, or 10 µg/mL anti-CD300a , or 0.1, 1, or 10 µg/mL anti-Siglec-8  for 45 minutes before measuring beta-hex release via supernatant and cell lysate as described above.A colorimetric assay to detect beta-hexosaminidase (beta-hex) release was used as previously described  with mod6 cells/mL). Cells were incubated overnight with peanut IgE mAbs  or Der p 2 IgE mAbs , followed by 24-hour stimulation with buffer, peanut, Der p 2, or Ara h 2 antigen. At 24 hours, supernatants were harvested for ELISA or Luminex. GM-CSF was measured using ELISA kits from Abcam . IL-13 was measured using Rat Luminex Discover Assay  according to the manufacturer’s protocol.24-well flat bottom plates were seeded overnight with 500 ul of RBL SX-38 cells (1.2 x 106 cells/mL). Cells were washed once with NaPIPES buffer, and then stimulated for either 1 hour or 4 hours with 1 µg/mL peanut, Ara h 2, or Der p 2 antigen. qPCR was performed as previously described in . Primer details below:RBL SX-38 cells were sensitized with 1 µg/mL peanut IgE mAbs  or Der p 2 IgE mAbs  overnight in 12 well plates  and presented as mean ± SEM. The two-way ANOVA test was used to compare degranulation of peanut and Der p 2 sensitized cells stimulated with antigen. The Kruskal-Wallis test was used to compare different experimental groups from remaining experiments. Kruskal-Wallis analysis did not include correction for multiple comparisons. P values <0.05 were considered statistically significant.The original contributions presented in the study are included in the article/JS contributed to the study conception and design, data collection, analysis and interpretation of results, and manuscript preparation and review. OI contributed to study design, interpretation of results, manuscript preparation and review. YZ, PY, and RG contributed to data collection. MK contributed to interpretation of results and manuscript review. SS contributed to reagent development, interpretation of results and manuscript review. AB contributed to manuscript review. All authors contributed to the article and approved the submitted version.This work was supported by the NIH/NIAID K08AI141691 (OI)); and R01AI155668, R01AI130459, and R21AI123307 (to SS); a 2020 American Association of Allergy, Asthma, and Immunology Foundation Faculty Development Award (OI); the National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, through Grant Award Number UL1TR002489; the North Carolina Biotech Center Institutional Support Grant 2017-IDG-1025, and the National Institutes of Health 1UM2AI30836-01. MK receives funding from National Institutes of Health and Department of Defense. JS was supported by the Initiative for Maximizing Student Development (NIH grant R25GM055336). AB receives grant support to his institution from the National Institutes of Health and the Burroughs Wellcome Fund. The UNC Flow Cytometry Core Facility is supported in part by NIH P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center. The UNC Advanced Analytics Core is supported by NIH Grant P30 DK034987. Research reported in this publication was supported in part by the North Carolina Biotech Center Institutional Support Grant 2017-IDG-1025 and by the National Institutes of Health 1UM2AI30836-01. The funders had no role in the analysis, decision to prepare or to publish, the manuscript. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.We thank the UNC Flow Cytometry Core. We thank Dr. J.P. Kinet for approval to use the RBL SX-38 cells and Dr. Michelle Hernandez for providing us with the RBL SX-38 cells. We also thank Dr. Soheila Maleki for purified Ara h 2, and Johnny Castillo Cabrera and Dr. Albert Baldwin for technical assistance with western blot imaging. We also thank Carlton Anderson of the UNC Advanced Analytics Core. Lastly, we thank Drs. Eveline Wu and Adam Leitzan for critical review of this manuscript.SS is an inventor on a patent  describing a method to make IgE monoclonal antibodies and receives royalties for intellectual property licenses with Indoor Biotechnologies. OI is a consultant for Blueprint Medicines and Novartis. MK receives consulting fees from Ukko Inc. AB receives royalties from UpToDate; consulting honorariums from Astella Pharma Global Development, Allergy Therapeutics (UK) Ltd, DBV Technologies, kaléo, N-Fold, LLC, ALK-Abelló, Inc and UKKO, Inc. as well as Aimmune Therapeutics, Consortia TX, Inc., and Prota Therapeutics for his service on their respective Scientific Advisory Boards. AB owns stock in Allertein and Mastcell Pharmaceuticals.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Malassezia spp. The Acari Hypothesis posits that the mammalian innate immune system utilizes pathogen-bound acarian immune effectors to protect against the vectorial threat posed by mites and ticks. Per The Hypothesis, IgE-mediated allergic disease is a specious consequence of the pairing of acarian gastrointestinal materials, e.g., allergenic foodstuffs, with acarian innate immune effectors that have interspecies operability. In keeping with The Hypothesis, the IgE profile of atopic patients should include both anti-acarian antibodies and specious antibodies responsible for specific allergy. Further, the profile should inform on the diet and/or environment of the acarian vector. In this regard, the prevalence of Demodex and Dermatophagoides on the skin of persons suffering from atopic dermatitis is increased. Importantly, the diets of these mites correspond well with the allergens of affected patients. In this report, roles for these specific acarians in the pathogenesis of atopic dermatitis are proposed and elaborated.Atopic dermatitis is a chronic relapsing dermatopathology involving IgE against allergenic materials present on mammalian epithelial surfaces. Allergens are as diverse as pet danders, and polypeptides expressed by microbes of the mammalian microbiome, e.g.,  The Acari Hypothesis posits IgE-mediated immunity is intended primarily to protect mammals against pathogen-bearing acarians, i.e., mites and ticks. Further, it theorizes the means by which such adaptive responses occur ,2. ImporH2 cells, and infiltration and degranulation of eosinophils [H2 response [The Hypothesis expounds the benefit of IgE-mediated immunity as an intended anti-vector defense whilst anticipating the potential for immunopathology, a consequence of specious IgE against non-vector materials. The original proposal focused on anaphylaxis, the prototypical early phase allergic reaction, because that reaction is suited in both scale and locale to removal of invasive ectoparasites . The latinophils , seems eresponse . It is tThe late phase of allergy also aligns teleologically with an anti-acarian objective. If physical means do not remove the ectoparasite in rapid fashion, the local environment becomes toxic, thereby either eliminating the acarian or influencing its migration. The late phase of allergy, then, represents local escalation of the allergic process. Inasmuch as acarian-borne pathogens can kill mammalian hosts , reparatThe character of allergic inflammation depends upon the duration of allergen exposure: transient exposure elicits an early phase response; prolonged exposure includes a late phase response; and unremitting exposure yields chronic inflammation. In the case of foodstuffs and certain environmental allergens, e.g., peanuts and bee venom, removal of a limited amount of exogenous allergen is uncomplicated and the manifestations of exposure are singular and transient. Similarly, seasonal exposures to other exogenous allergens, e.g., pollens and/or mold spores, yield seasonal manifestations. However, the mammalian immune system also encounters persistent microbial antigens, e.g., skin flora. And, just as transient antigens incorporate erroneously into an anti-acarian response, so, too, do persistent ones, the failed clearance of which by IgE yields chronic disease.Malassezia [Malassezia IgE must then be a consequence of the affected individual having been exposed to an acarian harboring Malassezia materials within its gastrointestinal tract. Thus, the responsible acarian most likely consumes fungi as a part of its diet. Given that AD patients routinely produce IgE against the danders of cats and dogs, the culpable acarians likely either inhabit or regularly approximate mammalian skin, i.e., Demodex and/or Dermatophagoides.Atopic dermatitis (AD) is a chronic IgE-mediated inflammatory condition . As was lassezia ,11,12, tSarcoptes scabiei), and demodicosis (Demodex spp.) [H2-polarized inflammation [AD is a chronic relapsing dermatopathology that in developing countries now affects 10–20% of children and 1–3% of adults . As no dex spp.) ,18,19. Hammation ,22. Resiammation ,23,24,25ammation . Despite significant understanding of the cellular effectors and the cytokine pathways involved in AD, the pathogenesis of the condition remains poorly understood. In this regard, there are two competing hypotheses. One, the ‘inside-out hypothesis’, proposes aberrant IgE sensitization prompts inflammation that compromises the integrity of the epithelial barrier. The other, the ‘outside-in hypothesis,’ proposes disruption of the epithelial barrier enables penetration of allergenic materials into the skin, resulting in immune activation ,28. As lAtopic march refers to the sequential progression of atopic manifestations that occurs in a subset of AD patients ,32. MultThe Acari Hypothesis accounts for AD, atopic march, and related phenomena altogether differently. According to it, the immune system of an individual with AD is not dysregulated. Instead, that immune system is responding as intended to a vector-active acarian. Having paired with an acarian pattern recognition receptor, dietary elements—even materials from human skin flora—of the culprit mite or tick are perceived by a human recipient as acarian in nature, subsequently eliciting ‘specious’ IgE and its attending inflammation. Atopic march is then attributable to persistent or repeated infestation by a vector-active acarian, not to compromised barrier integrity/function.H2-polarized response in leukocyte-specific fashion. In mast cells and basophils, allergen-induced crosslinking of bound IgE elicits release of pre-formed inflammatory mediators, e.g., histamine [H cells along the TH2 differentiation pathway, amplifying the TH2 response [D. pteronyssinus, cleaves CD23 from the surface of human B cells [IgE is uniquely mammalian, and all extant mammals express it. It initiates and directs atopic inflammation, including AD . In humaistamine . Dendritistamine ,42, and istamine ,44. Actiresponse . Interesresponse , a measuresponse . Importa B cells . Whether B cells ,50 argueDemodex spp. [H2 response by mobilizing the same cells and eliciting the same cytokines as IgE [IgE is central to atopy and a clear link between IgE and AD exists; however, the exact role of IgE in AD remains unsettled. The reason is that ~20% of patients with AD do not have elevated levels of either total or specific IgE . This, idex spp. . Chitin s as IgE ,56. WhatMalassezia stand out. For this reason and because their roles in AD have been proposed and elaborated [Malassezia are discussed next.Recognition of the centrality of IgE in the pathophysiology of AD has prompted many attempts to characterize the entirety of IgE allergen profiles ,59,60,61aborated , MalasseMalassezia are lipid-requiring basidiomycetes central to the pathophysiology of some forms of AD. Although healthy persons are not usually sensitized to Malassezia, many AD patients are, as demonstrated by patch tests, skin prick tests and/or the presence of Malassezia-specific IgE [Malassezia-specific IgE [Malassezia lack a fatty-acid synthase gene, they require for survival an exogenous source of fatty acids [Malassezia localize to sebum-rich sites, including the scalp, face, chest and upper back [Malassezia predominate, e.g., M. restricta preferentially colonizes the forehead and external auditory canal whilst M. globosa colonizes the back and occiput [ific IgE . Indeed,ific IgE . As mostty acids . In the per back . Across  occiput .Malassezia is associated with a subtype of AD that is limited to the head and neck [Malassezia in 100% of patients with such limited AD, but in only 28% of patients with AD that lacked head and neck involvement [Malassezia, and (2) anti-fungal therapy alleviates disease [Malassezia are claimed cardinal to development of the AD subtype. Inasmuch as complexation of Malassezia allergens with IgE can induce inflammation characteristic of AD, it seems reasonable to assume that, in this case, IgE is the primary effector of disease.Interestingly, IgE against and neck ,58,65,66olvement . For thi disease , MalasseMalassezia involvement in AD helps account for many other AD-related phenomena. As one example, alkaline soaps elicit AD lesions in susceptible individuals [Malassezia allergens [M. globosa analog of heat shock protein 70 (hsp70) is IgE-reactive in some AD patients [ividuals . Alkalinllergens . Relatedpatients . In fungpatients . Thus, iMalassezia involvement in AD is best supported, it is conceivable that any skin microbe might express proteins capable of eliciting anti-acarian responses. Indeed, IgE against staphylococcal allergens have been demonstrated in AD patients [Malassezia, other patterns of clinical AD can likely be attributed to other skin flora. Although Malassezia are the primary fungal organisms of adults, the fungal organisms of children are much more diverse [Although patients ,60. Just diverse , potenti diverse .Demodex and Dermatophagoides, have been demonstrated in increased number on the skin of AD patients [Demodex spp.  are ectoparasites specially adapted to inhabit mammalian skin. They reside within the pilosebaceous unit [D. folliculorum, which resides within hair follicles, and D. brevis, which resides within sebaceous glands [Demodex derive sustenance from sebum primarily, and from the contents of follicular and glandular epithelial cells [Demodex routinely colonize the face, ears and neck, and less commonly colonize the chest, back, genitals and buttocks [Demodex are found within ectopic sebaceous glands of buccal mucosa [Importantly, two genera of acarians, patients ,76. Demoous unit . Over 10s glands . Demodexal cells ,79. Consbuttocks . Demodexl mucosa  and withl mucosa . Demodex colonize both humans and domesticated mammals [Demodex spp. are mostly monoxenic, human infestation by non-human species occurs [D. folliculorum and D. brevis, have been isolated from privately owned cats [Demodex betwixt pets and humans occurs. Additionally, because Demodex feed on epithelial materials of their hosts, interspecies migration likely facilitates generation of specious IgE against cat and/or dog dander.  mammals . Althougs occurs ,84. Furtned cats . Thus, mDemodex spp. contributes to at least one human disease. Rosacea, a chronic, relapsing dermatopathology of the face, is characterized by demodicosis and elevated IgE [Bacillus oleronius, isolated from Demodex [B. oleronius, it has been hypothesized that this mite-derived bacterium is cardinal to pathophysiology [There is evidence that the vector activity of ated IgE . Importa Demodex . As the ysiology . D. folliculorum populations that inhabit family units and ancestral human lineages are informative. Firstly, the sharing of D. folliculorum haplotypes within families indicates that frequent close physical contact leads to transmission of mites. Secondly, ancestral human lineages maintain their mite pedigrees, e.g., African Americans maintain an African mite pedigree whilst European Americans maintain a European mite pedigree [Phylogenetic analyses of pedigree . The impMalassezia, Demodex subsist on sebum [M. furfur, lipases are active between pH 4 and 10 [Demodex infestation results in increased pH of afflicted skin [Demodex likely contains malassezial elements.Like on sebum , and boton sebum ,90. This4 and 10 . Althoug4 and 10 . Such prted skin . Thus, iDemodex, Dermatophagoides  are strongly associated with AD and patients with AD produce anti-Dermatophagoides antibodies [Dermatophagoides do not consume sebum. Their diet consists primarily of mammalian dander. Additionally, they are mycophagous and consume Alternaria alternate and Cladosporium sphaerospermum [Malassezia to Dermatophagoides. Given the presence of the latter on human skin and the paucity of non-malassezial fungal species, ingestion of Malassezia by Dermatophagoides seems likely. In contrast to tibodies . Dermatoospermum , well-knospermum ,96. No sIn keeping with The Acari Hypothesis, AD is a disease process attributable to either: (1) active epithelial infestation by an acarian, or (2) an immunopathological state elicited by an acarian infestation, during which bystander materials were inadvertently targeted. Given either of these, initial management of AD should include identification and elimination of the acarian. Further symptom mitigation would then involve delineation of relevant allergens, including and especially microbes and their associated antigens. Having delineated relevant allergens, clinical disease might best be ameliorated using therapies that minimize allergen burden and/or disrupt relevant immune cascades. Allergen burden might be eliminated by avoiding environmental triggers that prompt microbial expression of relevant allergens  and/or reducing the number of allergen-producing microbes . Symptom-inducing immune cascades might be interrupted using: (1) omalizumab, a monoclonal antibody that inhibits the binding of IgE to FcεRI ,98, (2) Rhipicephalus appendiculatus, might reduce AD-associated itch and pain [As targets of IgE, acarians have evolved means to neutralize or evade the effects of IgE activation. Thus, agents derived from the acarian proteome might prove therapeutic. As one example, votucalis, a histamine-binding protein expressed in the saliva of the tick, and pain .Conceptually, IgE should be regarded as either an anti-acarian immune reactant or as a specious—albeit acarian-triggered—immunopathological reactant. Gross responses elicited by IgE, i.e., itching, spitting, vomiting and defecating, operate as intended to rid epithelial surfaces of what the mammalian immune system recognizes/perceives as vector-active acarians. The prevalence of IgE against synanthropic acarians suggests the vector activity of the organisms must be especially problematic for mammals. Given the narrow functionality of IgE and its conservation across all extant mammalian lineages, it seems likely that one or more acarian species exerted selective pressure critical to the evolution of class Mammalia.Demodex have been implicated in the pathophysiology of recurrent herpetic keratitis [Demodex colonization, it is plausible that Demodex plays a role in all forms of recurrent herpetic infection.Although this report elaborates a role for acarians in the pathophysiology of AD, other diseases associated with IgE likely also involve acarians. eratitis . Personseratitis , a findieratitis . Of noteeratitis . Given: Chronic obstructive pulmonary disease (COPD) is a relapsing condition characterized by poorly reversible obstruction of pulmonary airways and by pulmonary inflammation . A hallmMycobacterium leprae, involves the skin and peripheral nerves, especially those of the face and hands [Dasypus novemcinctus, is a reservoir of M. leprae, and persons that spend time in the habitat of D. novemcinctus are at risk for contracting leprosy, even without directly contacting armadillos [M. leprae. Indeed, at least one tick, Amblyomma maculatum, can harbor M. leprae and transmit it vertically [A. maculatum can also affect horizontal transmission of M. leprae to vertebrate hosts [Demodex may serve as bacterial vector [M. leprae have elevated IgE [Leprosy, a chronic infectious disease caused by the obligate intracellular bacterium, nd hands . Althougmadillos . For thirtically . A. macute hosts . Human-tl vector ,119. Related IgE ,121, conAlthough only hypothetical, these examples suggest acarians may be influencing human health in ways not previously considered. The Acari Hypothesis provides a plausible contextual framework with which to test these and other proposals relevant to IgE-mediated diseases, not the least of which is atopic dermatitis.


Jupiter’s moon Europa is a prime candidate for extraterrestrial habitability in our solar system. The surface landforms of its ice shell express the subsurface structure, dynamics, and exchange governing this potential. Double ridges are the most common surface feature on Europa and occur across every sector of the moon, but their formation is poorly understood, with current hypotheses providing competing and incomplete mechanisms for the development of their distinct morphology. Here we present the discovery and analysis of a double ridge in Northwest Greenland with the same gravity-scaled geometry as those found on Europa. Using surface elevation and radar sounding data, we show that this double ridge was formed by successive refreezing, pressurization, and fracture of a shallow water sill within the ice sheet. If the same process is responsible for Europa’s double ridges, our results suggest that shallow liquid water is spatially and temporally ubiquitous across Europa’s ice shell. The formation of double ridges on Europa is poorly understood. Here the authors analyze airborne radar observations of an analog feature on the Greenland Ice Sheet to show that the refreezing of shallow water sills may produce such ridges. The thickness and thermophysical structure of this ice shell are poorly constrained, but models suggest it may be 20–30 km thick8 with a layer of warm, convecting ice underlying a cold, rigid crust10. The detailed structure and dynamics of its ice shell and the timescales over which they evolve are critical for understanding both the fundamental geophysical processes and habitability of Europa11. Some of the primary observational constraints on these subsurface processes are their expressions in the surface morphologies imaged by Voyager and Galileo.Jupiter’s icy moon Europa harbors a global subsurface ocean beneath an outer ice shell12 and geologically active14, displaying a wide variety of landforms including ridges, troughs, bands, lenticulae, and chaos terrain15. Of these, double ridges are the most common, consisting of quasi-symmetric ridge pairs flanking a medial trough16, with height to peak-to-peak distance ratios <0.5817. These ridges may extend for hundreds of kilometers and include some of the oldest features visible on the surface, with frequent cross-cutting implying numerous formation cycles over Europa’s history16. Cycloidal ridges and ridge complexes share many of these characteristics and along-strike transitions between ridge morphologies are not uncommon, suggesting that a single process may be active in the formation of all ridge types19. Proposed formation mechanisms for double ridges fall into six categories: cryovolcanism20, tidal squeezing13, diapirism21, compression22, dike intrusion or ice wedging24, and shear heating26. All of these mechanisms require ice-shell fracture and, with the exception of compression and diapirism, all invoke near-surface ice-water interactions, either through internal melting26 or direct injection from the subsurface ocean24.Europa’s surface is young27. These models suggest that double ridges may instead form above shallow, crystallizing water bodies, such as sills or dikes, within the ice shell, rather than by direct connection to the subsurface ocean30. Such mechanisms are more consistent with morphometric analyses that both disfavor compressional formation models and support the presence of subsurface water reservoirs beneath ridges28. These models have much in common with formation mechanisms for lenticulae, chaos, and cratered terrain that invoke the emplacement and refreezing of shallow water bodies to explain the observed doming, surface disruption, or collapse33. This growing body of work suggests that shallow water may be critical, not only to double ridge formation, but also to Europan ice-shell dynamics, exchange, and ultimately habitability.More recently, a number of extensions to the explosive cryovolcanism hypothesis have been proposed, in part to address the difficulty of driving negatively buoyant ocean water directly to the surface34. Sea-ice pressure ridges35 and some ice rise divides36 bear a qualitative resemblance to Europa’s double ridges, but the pressure ridge analog assumes a very thin ice shell and ice divides express a flow regime entirely dissimilar to the Europan environment. Similarly, subglacial volcanic craters and ice shelf brine infiltration have been invoked as analogs for pressurized ice-water interactions32, but neither fully captures the physics of refreezing water bodies confined within an ice matrix.Analogs for double ridges or confined shallow water bodies from the terrestrial cryosphere could place powerful constraints on this hypothesis space for EuropaHere, we present an icy double ridge discovered on the Greenland Ice Sheet with the same gravity-scaled geometry as Europa’s double ridges Fig. . High-re17. Surface imagery and the DEMs also show evidence of flexure along the ridge flanks , or smaller ice lenses (<0.1 m thick) with limited horizontal extent. Previous work has shown that the subsurface structure in this area typically consists of a 3–5 m thick low-permeability ice slab perched above a partially porous firn layer with significant ice lensing that transitions to solid ice at a depth of 30–40 m37, suggesting that it was originally emplaced within the relict porous firn layer. However, a localized surface uplift of up to 9 m associated with sill emplacement . The sill is surrounded by an ellipsoidal region of extremely low relative radar reflectivity and minimal stratigraphic structure, consistent with a homogeneous region with minimal internal contrast in material density  Multichannel Coherent Radar Depth Sounder (MCoRDS) reveal that this double ridge is underlain by a shallow water sill  and atmospheric pressure (0.1 μPa) at Europa should aid ridge formation. Both conditions act to reduce overburden on the sill, relaxing the overpressure conditions needed to drive water towards the surface or uplift the material overlying the sill. A smaller fraction of the sill might be required to refreeze to form the ridges in this case, but only if the degree of refreezing needed to achieve the minimum overpressure exceeded the degree of refreezing needed to generate the added ridge volume by expansion.40, and most models of sill emplacement on Europa have assumed depths 1–5 km for sill intrusions31. This would place sills firmly within the brittle, conducting portion of the ice shell where temperatures likely do not exceed 150 K (ref. 8), except where latent heating can warm the surrounding ice. Therefore, we expect elastic deformation or brittle fracture to dominate in response to imposed stress, with little to no contribution from viscous creep. This is consistent with our proposed mechanism, which invokes fracture and rapid uplift or extrusion of damaged material to form the ridges, despite the significantly warmer conditions in Greenland (~260 K). However, the length scales and timescales for ridge formation would be strongly modulated by the details of the rheology.Ice temperature and chemistry are poorly constrained confounding factors at Europa that play an important role in governing ice rheology. Our model invokes some degree of near-surface porosity, suggesting that sills would need to form within or at the lower boundary of the porous portion of Europa’s ice shell. Previous work estimates this near-surface layer to be on average 3 km thickThese temperature differences also imply an absence of surface melting on Europa, whereas melt is common at the double ridge site in Greenland. In particular, Landsat 7 ETM+ and Landsat 8 OLI optical satellite imagery show visible surface runoff and localized water ponding within the basin during four out of the five summers spanned by our DEM and radar observations  to characterize the evolving surface morphology of the double ridge between 15 April 2012 and 25 March 2016. The strip-map ArcticDEM products are stereo-photogrammetric DEMs with 2 m posting produced from satellite image pairs collected by the WorldView-1, WorldView-2, and WorldView-3 satellites. We select DEMs with quality flags that exceed 0.98 (out of 1). The DEMs are provided with precalculated offsets to the x, y, and z spatial coordinates determined by co-registration to ICESat laser altimetry tracks, which we apply to the data to reduce absolute positioning errors in all three dimensions. The median relative vertical uncertainty is estimated to be on the order of 20 cm for these products after co-registration49. We measure the ridge surface morphology (ridge heights and peak-to-peak distances) from the 25 March 2016 DEM.We use strip-map ArcticDEM17. To compare the geometry of the Greenland ridge to those on Europa, we calculate this same ratio for the terrestrial double ridge. The measured ridge height is 2.1 ± 0.57 m, with the northern ridge ~1.4 times the height of the southern ridge. The peak-to-peak distance varies from 26 to 78 m along-strike, with an average width of 46 m. We scale the measured height of the ridge by the ratio of the gravitational acceleration on Earth to that on Europa (ratio of 7.45), as any ridge-building mechanism would require uplift in direct opposition to this force. After scaling, we find a height to peak-to-peak ratio of 0.37 ± 0.17 (μ ± σ), which falls comfortably within the range of values calculated for Europa’s ridges17.Coulter, et al. (2009) demonstrated that Europa’s ridges have a consistent relationship between ridge height and width, characterized by the ratio of the mean ridge height to peak-to-peak distance50 operating at a center frequency of 300 MHz with 300 MHz of bandwidth, giving a range resolution of 0.42 m in solid ice and 0.51 m in firn (assuming a firn refractive index of 1.5). These data were focused using F-K migration with a 2.5 m target azimuth resolution. As displayed in Fig. We use radar sounding data from two different systems flown as part of NASA’s Operation IceBridge and built by the University of Kansas Center for the Remote Sensing of Ice Sheets (CReSIS). Deep sounding data were collected on 10 May 2016 using the Multichannel Coherent Radar Depth Sounder (MCoRDS)51. In 2015 and 2016 this system operated at a center frequency of 5 GHz with 6 GHz of bandwidth for a vertical resolution of ~2.2 cm and ~3.8 cm in ice and firn, respectively. These data were coherently averaged 32 times and incoherently averaged 11 times with a moving window before decimation by a factor of 5 to give ~10 m along-track resolution and ~5 m trace spacing. In 2017, the system operated with a center frequency of 10 GHz and 16 GHz of bandwidth for a vertical resolution of ~0.9 cm and ~1.4 cm in ice and firn respectively. These data were coherently averaged 32 times and incoherently averaged 11 times with a moving window before decimation by a factor of 5 to give ~10 m along-track resolution and ~5 m trace spacing.The time series radargrams in Fig. With the exception of the 2015 Snow Radar data, all radargrams were elevation corrected using the radar-derived flight clearance and aircraft altitude from the system GPS and assuming an index of refraction of 1.78 (solid ice) in the subsurface. Due to significant along-track positioning errors in the 2015 Snow Radar GPS data, we use the 2015 ArcticDEM surface elevation extracted along the flight line for elevation correction in this season.52. To find the system calibration constant, we cross-level this flight line with two interior tie-lines that intersect the sites of the B2953 and B1654 firn cores of the North Greenland Traverse. We then absolutely calibrate the data against simulated subsurface reflectivities using high-resolution density measurements from these cores55. We correct for englacial attenuation using the average firn conductivity from the B26–B29 firn cores59 and an englacial temperature profile60 simulated by the regional climate model MARv3.5.261. Details of these correction and calibration procedures and associated sensitivity tests are described in the 62, which is unlikely to hold for radars with centimeter-scale wavelengths.We absolutely calibrate the 2016 MCoRDS radar data by correcting the received radar power for geometric spreading loss, system offsets, and englacial attenuation. We geometrically correct the data using a range squared dependence63. This distribution of observed reflectivities may already be an underestimate of the true permittivity contrast, as we observe significant off-nadir returns that suggests that some power was lost to radar scattering at a rough ice-water interface, although our synthetic aperture processing partially mitigates this effect63. The sudden disappearance of otherwise conformable englacial stratigraphy directly below this reflector in the 2017 MCoRDS data suggests significant attenuation of the radar waves as well. To estimate attenuation, we select five englacial reflectors located 100–200 m below the water table and compare their average power over 1 km segments to the left and right of the water table with the average received power directly beneath the water table at the equivalent depth. We observe an average power reduction of ~11 dB beneath the water table, relative to the surrounding ice. Assuming that the bottom of the sill extends no deeper than observed bottom of the refrozen ice shell, this implies an additional 11 dB of attenuation occurs over 15 m depth, equivalent to an englacial attenuation rate of 367 dB/km. Liquid water is the only reasonable mechanism for this absorption loss, which is almost 22 times greater than the typical depth-averaged englacial attenuation rates of 15–20 dB/km in this region64.The presence of subsurface liquid water is the only reasonable interpretation for the observed reflectivity and attenuation signals of the interface we identify as a water table. This interface has a calibrated reflectivity of −12.1 (7.468) dB (median and interquartile range), with 62% of the observed values falling between the theoretical reflectivity of a specular interface between ice and saturated firn (−14 dB) and the theoretical reflectivity of a specular ice-water interface (−3.5 dB)6 m3 void could be formed and maintained for years without surface collapse, and ~41% of the reflectivity observations actually exceed the theoretical reflectivity of a specular air-ice interface. Additionally, void space could not produce the observed attenuation beneath this feature.The only other physical configuration, which could plausibly produce the observed mean reflectivity, would be if the subsurface contained a massive air-filled void, rather than a water body. However, it is physically unlikely that a 7.2 × 1066, we estimate the initial sill water volume based on the surface displacement. We measure the surface displacement associated with sill emplacement by differencing the co-registered DEMs collected on 15 April 2012 and 9 July 2013. We estimate the uncertainty in absolute elevation change by adding in quadrature the 70th percentile of the absolute vertical deviation from the ICESat registration track  for each DEM as provided with the product registration files. The absolute uncertainty in surface elevation change for this DEM pair is estimated to be ±1.18 m. We manually select the bounding outline where the minimum surface displacement occurred and discretely integrate the surface elevation change within this boundary to estimate the displaced surface volume. We estimate uncertainty by calculating the total displaced volume assuming the surface displacement was either 1.18 m greater or less than measured. This volume estimate is likely an underestimate as it does not account for leak-off into the surrounding firn pore space. This method results in an estimated initial sill volume of 7.2 × 106 ± 7 × 106 m3.Assuming that the displaced surface volume is equal to the injected subsurface water volume for sill-like features67. The observed maximum uplift of 9 m is far in excess of the annual accumulation simulated by MARv3.5.2 in this region, which is on the order of 1.2 m assuming a snow density of 0.3 gcm−3 and no compaction or melt. Ice flow also is unlikely to produce this kind of singular, localized uplift over a horizontal length scale less than the mean ice thickness, particularly as surface basins are understood to be expressions of stable subsurface topography.We assess that subsurface water injection is the most likely source of the observed uplift as we observe a subsurface water reservoir and the uplift occurred during or immediately after the extreme melt season in summer 20125 m3.We estimate the total volume of the ridge from the 2016 DEM. We extract the portion of the DEM within 500 m of the outer edges of the ridge system, excluding the region occupied the ridge. We fit a mean plane to this surface, which we subtract from the full DEM to produce a map of local elevation relative to the mean surface of the basin. We then integrate the total volume within the area manually delineated as containing the double ridge to determine a total volume of 1.2 × 1055. Based on the horizontal radar coherence and field observations from Greenland’s ice slab areas69 . A single realization of footprint effective reflectivity is then calculated by randomly choosing the number and area of firn patches such that they cover the desired fractional firn area, generating a random vertical density profile for each firn patch based on the model parameters, simulating the effective reflectivity of each firn patch with a 1D layered dielectric reflection model71, and coherently summing the reflectivity of all patches. The To test our hypothesis that the sill is encased in a refrozen ice shell, with concentrated water injection and refreezing directly beneath the ridges and medial trough, we invert the observed radar reflectivity in each region for fractional ice content using an electromagnetic forward model that parametrizes radar reflectivity in terms of the density and geometry of the subsurfaceas69 see , we modeWe establish distributions of radar reflectivity associated with the firn, refrozen ice shell, and sub-ridge material using the SAR-focused 2016 MCoRDS radargram. Supplementary Figure Fice, from the estimated model parameters following equation (1), where Vradar is the volume of the illuminated radar footprint, Afirn is the fractional surface area of that footprint containing layered firn, and MFP is the fractional height of ice layers within those firn patches.As our forward model is both non-linear and stochastic, we use the Markov Chain Monte Carlo (MCMC) method with a Metropolis-Hastings sampling scheme for inversion. This allows us to estimate full empirical distributions of model parameters that are consistent with the observed radar reflectivity distributions. Supplementary Table We reprocess the 2016 Snow Radar data without any along-track averaging  in order to emphasize hyperbolic scattering from point features and rough surfaces. This data, with an along-track trace spacing of ~0.15 m, is shown in Supplementary Fig. 68 suggest that this is a sufficiently shallow depth that these features would be subject to seasonal temperature variations in response to changing atmospheric temperatures. For liquid water to persist at these shallow depths over winter would therefore require a sufficiently large water volume that latent heat exchange could prevent complete refreezing. However, the thermal profile of this area is likely complicated by seasonal surface ponding and ongoing refreezing of a large subsurface water body, which might result in warmer firn at shallower depths and make shallow water storage more viable than typically assumed. On the other hand, both surface and snow bridged crevasses commonly produce similar signatures in ice penetrating radar data, either due to diffraction where the fracture plane meets the surface, or scattering from irregularly shaped snow caps over subsurface void space72. Therefore, we interpret these returns as most consistent with scattering from some form of void space beneath the central trough. This could include scattering from different corners of an irregularly shaped void over a single conduit that was not fully filled by pressurized water, or diffraction at the tips of multiple adjacent, narrower fractures. Regardless, even in the case that liquid water is present, the distinct and separable nature of the hyperbolic returns suggests that we observe discrete, isolated liquid water pockets rather than a broad region of saturated pore space. This similarly supports our interpretation of fracture development beneath the medial trough.The presence of liquid water in late spring at these shallow depths is less likely than void space due to thermal constraints. The radar data we analyze were collected in May 2016, just prior to the onset of the melt season, so any liquid water would have needed to survive the preceding winter without refreezing. The observed scatterers are located ~2–4 m below the surface, and firn temperature profiles from ice slab regions in Southwest Greenland73 flow accumulation analysis to the 32 m ArcticDEM surface topography product to confirm that the ridge is located within a closed basin with no route for surface water outflow in all years from 2012 to 2016. In April 2012, prior to the observed surface uplift, a lake depth of roughly 14 m at the center of the depression would have been required to overtop the basin based on ArcticDEM strip DEMs. Landsat 7 ETM+ optical imagery from the summers (June/July/August) of 2009, 2010, and 2011 show that a small surface lake formed in this basin during each melt season, likely through a combination of local melt and lateral runoff from upslope ablation (Supplementary Fig. We apply the TopoToolbox67, with record-breaking surface runoff61 and bare ice exposure74 in this region. Based on these observations, we infer that the majority of the surface water that flowed into this basin in the summer of 2012 drained into the subsurface through fractures in the overlying ice slabs, leading to the observed surface uplift and apparent absence of ponded surface water, while forming the sill we later observe in the 2015 radar data.Between April 15, 2012 and July 9, 2013, this basin experienced a surface uplift of up to 9 m (Supplementary Fig. Supplementary InformationPeer Review File


A 3D icing simulation code is developed in the open-source CFD toolbox OpenFOAM. A hybrid Cartesian/body-fitted meshing method is used to generate high-quality meshes around complex ice shapes. Steady-state 3D Reynolds-averaged Navier-Stokes (RANS) equations are solved to provide the ensemble-averaged flow around the airfoil. Considering the multi-scale nature of droplet size distribution, and more importantly, to represent the less uniform nature of the Super-cooled Large Droplets (SLD), two droplet tracking methods are realized: the Eulerian method is used to track the small-size droplets (below 50  When an aircraft flies through a low-temperature cloud that is rich in moisture, the supercooled droplets in the cloud will impinge the aircraft surface, probably accompanied by ice accretion through mass and heat exchange phenomenon . The airIn the last two decades, 2D and 2.5D in-flight ice prediction codes have been developed and widely used by the aerospace industry. Sometimes, wind tunnel tests can engage scaling issues if sub-scale models are used or blockage and configuration problems if full-scale models are used where full configuration and full-scale effects are important. However, the 2D numerical methods cannot be employed on 3D geometries such as multi-element wings, nacelles, or engines, as well as integrated systems or situations that combine external and internal flows. Obviously 2D icing simulation does not take into account corrections for the sweep, up-wash, down-wash, twist, etc., which may lead to inaccuracy in predicting ice shapes.Many 3D icing simulation codes are costly and complicated, mainly in generating 3D mesh and obtaining the airflow solution, tracking droplets’ trajectories, and calculating ice accretion. For mesh generating, it has been revealed that high-quality mesh deformation techniques are essential to achieve a robust, accurate, and efficient process of flux calculations, especially viscous fluxes near the solid wall boundaries . The demFurthermore, the traditional difficulties encountered by the Lagrangian method include long calculation times, difficulty in determining impingement limits, sparsely seeded particles not covering complex surfaces, etc. For the ice accretion model, the traditional Messinger model  lacks thThis paper aims to provide a 3D icing simulation code implemented in OpenFOAM , which iThe whole process of the icing simulation is based on time marching to accumulate ice. Within each time step, the following four steps are executed sequentially: (1) calculation of the aerodynamic flow field, by neglecting droplets’ effect on the airflow; (2) tracking the droplets’ trajectories due to aerodynamic force and computing the collected water mass on the aircraft surface; (3) an inner loop to add the droplet mass to the water film, solve the water film flow, and then estimate ice accumulation via heat transfer analysis; (4) generating the body-fitted mesh with ice accretion covered on the surface. The relation between the four steps and the outer time step loop is shown in Before diving into the numerical methods and physical models, we want to emphasize that aircraft icing, which involves complex flow, heat transfer, and phase change, is intrinsically a complicated thermodynamic phenomenon. The simulated physical process that results in ice accumulation, is irreversible. For example, the PDEs we solve for the air and water film flow, are convection-diffusion equations. The viscosity and diffusivity in the momentum and heat equations are always positive. Obviously, one can not solve the convection-diffusion phenomenon backward in time. In addition to the PDEs, some approximations and empirical models applied are also irreversible: in the Lagrange tracking method, a droplet that has a Weber number higher than a certain threshold is subject to splashing when approaching airfoil surface; in the icing model, the kinetic energy of the flying droplets is converted into the thermal energy of the water film; the Myers model, which provides an empirical solution to the 1D Stefan problem along the wall normal direction, is also irreversible in time. Overall, the nature and computer simulation of ice accumulation over time, is directed by the second law of thermodynamics, which cannot be reversed.Traditional finite-difference and finite-volume CFD methods rely on a structured body-fitted mesh to accurately compute the near-wall gradients. Indeed, body-fitted mesh allows accurate computation of wall stresses and heat fluxes. Note that the mesh has to be regenerated at each icing step due to the change of boundary shape which requires an automatic mesh generation method. Methods using structures mesh can stretch the mesh to fit the ice shape while keeping the topology unchanged. However, 3D ice accumulation can become fairly irregular, and significant deterioration of near wall mesh quality can be observed. Artificial smoothing on the ice shape may be necessary for structured mesh, which will certainly sacrifice the accuracy on the predicted ice shape for the need of computation robustness.To overcome this, a mixed Cartesian/body-fitting mesh, as shown in yHexMesh  where usThe mesh convergence study on the lift coefficient was performed for the NACA 0012 airfoil at the AOA of cCroskey . The simOne may point out that traditional mesh sensitivity study on aerodynamic simulations involves s LEWICE  icing soAccording to the authors’ experience, the water film is less sensitive to the grid resolution. Due to the extra refinement of Cartesian mesh within the potential icing region, the ice shape does not show much sensitivity to the grid resolution.Due to the mass, momentum, and heat exchange between the air and ice/film, the aerodynamic and temperature fields of the flow around the airfoil are prerequisites for solving ice accretion. The thickness of the water film is neglected here because of its insignificance relative to the airfoil size. Because the icing time scale can be in minutes which is significantly larger than the flow (turbulence) time scale, thus the airflow is assumed to be steady within each icing step. It is a standard practice in many icing software. The steady solution of the ensemble-averaged airflow field around the airfoil is obtained by solving Reynolds-averaged Navier-Stokes (RANS) equations in OpenFOAM. The compressible Semi-Implicit Method for Pressure Linked Equations (SIMPLE)  method iT) model  are popuIn the airflow simulations, Dirichlet boundary condition is given for velocity, turbulent kinetic energy, eddy frequency, and temperature at the inlet. Zero gradient boundary condition is applied at outflow for the transported variables. Pressure is set to zero gradient at inflow and fixed at outflow. To accelerate convergence, the velocity field is initialized by a potential solver.Considering the mesh can be non-orthogonal, for the purpose of solver robustness, the gradients are evaluated using the least-squares approach and are limited to ensure that the face values obtained by extrapolating the cell value to the cell faces using the gradient are bounded by the neighboring cells minimum and maximum limits. The convection of velocity is discretized by a second-order linear upwind scheme, with an explicit correction based on the local cell gradient. The convection of turbulent kinetic energy and eddy frequency are discretized by the upwind scheme to ensure boundedness. The number of non-orthogonal corrections is set to 2 to account for the max mesh non-orthogonality no greater than 65.For clean airfoil and rime ice conditions, where no significant flow separation is present, the velocity residual converges below The method is established based on the following assumptions: (1) droplets do not collide with one another and they do not have mass, momentum, or heat exchange; (2) droplets are deemed as spheres in calculating aerodynamic forces; (3) large droplets may experience break-up before impacting the surface; (4) splashing is only considered for the large droplets; (5) only the drag, gravity, and buoyancy are considered as the forces of airflow exerted on droplets; (6) the effect of droplets on airflow is negligible. These assumptions are applicable under the condition that the volumetric fraction of droplets is low  of the droplet size distribution; L represents the characteristic length ; As mentioned, the small-size droplets can be viewed as a continuous phase. Moreover, the Eulerian method is more efficient than the Lagrangian method in solving concentration problems under steady-state conditions. Thus the Eulerian method is more suitable in calculating the collection of small-size droplets. The Eulerian formulation of the droplet impingement process yields a set of droplet-related continuity and momentum equations, which are introduced by Bourgault et al. . Since ρfollows,∂α∂t+∇·(α(αUd)=0,∂αUd∂t+∇α(αUd)=0,∂αUd∂t+∇α(αUd)=0,∂αUd∂t+∇αUnder the icing conditions, it is considered that the airflow and the droplet are one-way coupled, namely the airflow field affects the droplet field only since the volumetric fraction of droplets is too low  is marcposition . The metu, and t represent the liquid water content, the value of air speed in the far field and the tracking time respectively.After the droplet tracking process, the collection efficiency is then defined as:β=McollecThe film model used in this code is proposed by Bai et al. , which ch represent the film thickness and enthalpy respectively. The subscript s in the The governing equations of the film motion are given:∂ρδ∂t+∇s·ρδU,spl,∂ρδh∂t+∇sd by Bai , which iThe fundamental transport equations for the liquid film are solved on an extruded surface mesh. The extruded surface mesh is not necessarily connected to the airflow mesh in a geometric sense, as only boundary data exchange is needed between the two meshes. The mesh is discretized in both directions tangent to the surface, but is only one cell thick in the direction normal to the surface, as shown in Note that the water film is solved on a separate 2D mesh while the airflow is solved on a 3D mesh. A data exchange process is required to make the water film boundary conditions physically consistent. The top surface of the water film needs pressure, shear stress, heat flux information from the air field. Since the extrusion of mesh makes sure of face connectivity, thus a direct data mapping would suffice. The bottom surface of the water film uses a non-slip, fixed temperature boundary condition.The gradients inside the film mesh are evaluated using least squares approach, similar to the one applied in flow simulation. The convection of velocity, turbulent kinetic energy and eddy frequency are discretized by upwind scheme to ensure boundedness.The Myers model  is adoptThe heat flow balance equation is defined asQk+Qai+QlThe ice growth rate equation is defined as∂b∂t=M˙coIn the second stage, water flow occurs when the latent heat is not enough to convert all the water into ice, which brings runback water, and the icing type changes from rime ice to glaze ice. The glaze ice conditions correspond to a regime between ice and water, and the temperature of both phases is supposed to be at the melting point. The reason for this two-stage process is that this model requires good heat transfer between the substrate and the incoming droplets. Under this prerequisite, the initial incoming water must immediately adopt the subfreezing substrate temperature and then freeze because it has a nucleation site. Only when there is a sufficiently insulating layer and enough energy has been introduced into the system can water appear. The formed layer may be relatively thin, but the freezing of even a small proportion of droplets may provide sufficient latent heat for the remainder to heat up and stay liquid as runback water. This is a consequence of an assumption that the droplets are supercooled and the temperature of the substrate remains fixed and subzero.When the icing type turns to glaze ice, the heat flow balance equation is defined asQk+Qaf+QlThe ice growth rate equation is defined as follows:ρglf∂b∂t=For a smooth transition from the rime to glaze models, the ice height, film thickness, and growth rate must be continuous, and thus fined asBg=ki, which is naturally capable of simulating a 3D scenario. However, most experimental data available in the literature are airfoil icing measurement which are intrinsically quasi-2D or 2.5D, so the validation is limited to 2.5D in the current work. The purpose is to validate that the solver, developed on OpenFOAM, is capable of predicting accurate ice shapes.The mesh sensitivity study on the aerodynamic field is given in The results in The ability to accurately predict ice shape is validated on the NACA 0012 airfoil, with comparisons made to the ice shapes measured in experiments ,27. The c is the airfoil chord length. There are 50 layers in the spanwise direction. On the airfoil surface, the streamwise face length ranges from The size computational domain is The results are then calculated using both the Eulerian and the Lagrangian methods. Different types of ice shapes are calculated including rime ice and glaze ice with small-size droplets, and SLD ice accretion.The icing simulation conditions for the small-size droplets and SLDs are shown in The collection efficiency for case 1 is shown in The validation results of ice shape for cases 1 and 2 are shown in For case 1, the upper and lower limits where the droplets hit agrees with the experiment result well. However, for case 2, the upper limit is closer to the leading edge compared to the experiment. It is possible that the error is originated from the airflow computation considering the ice shape causes flow separation on the upper surface. The steady-state solution from the RANS equation is known to overpredict separation bubble size. For that reason, given the high efficiency of RANS, it is acceptable for engineering purposes.Different from cases 1 and 2, the droplet size in case 3 belongs to the SLD category. Since the large droplets (SLDs) are less uniform and are more likely to splash, the resulting distribution of collection efficiency should be less uniform than small droplets. Thus the Lagrangian method may be more appropriate than the Eulerian method. In our numerical simulations of case 3 using the Lagrangian method, the injection positions of the large droplets are randomly determined at the inflow plane. The distribution of collection efficiency on the airfoil surface is shown in The global view of ice shape and comparison with experiment measurement of case 3 are shown in The spanwise-averaged 2D ice shape b impliesIn summary, the icing simulation proves to be feasible under small-size droplet condition with the Eulerian method and SLD condition with the Lagrangian method. Characteristics of these two numerical methods of tracking and collecting droplets are demonstrated respectively. For the Eulerian method, the collection efficiency is calculated as a field, which is continuous in time and space, and this brings a clean ice shape as a result. This can be quite useful and time-saving when the body shape is complex. For the Lagrangian method, it is consistent that the droplet injection takes the randomness of droplets’ initial positions into account and the ice shape is more close to the experimental one with ice roughness generated, but this is time-consuming when the body shape is complex.Although there is a lack of trustworthy experimental data on realistic 3D geometries, the full 3D capability of the icing simulation code can still be demonstrated. The ONERA-M6 wing is studied in icing conditions. This wing is selected because it is a well-known geometry with several available experimental and numerical results for the airflow solution. It is often used for validation of numerical results in the transonic range, at high Mach number and high Reynolds number. In the context of ice accretion simulation, the Mach number and the Reynolds number have therefore been reduced.c is the chord length at wing root. Extra refinement of the volume mesh within the distance of c to the wing surface is applied. For this case, the number of cells is 1,630,000. The air flow field and ice shape does not show significant sensitivity to mesh resolution at this level. The mesh on the leading edge are refined to capture the 3D ice shape, as shown in The calculation conditions are shown in The predicted ice accretion on the M6 wing is shown in This case just proves the 3D icing simulation ability. Further complex 3D research can be performed via this code.In the above, the viability of developing a 3D icing solver based on the open-source platform OpenFOAM is demonstrated. The bottleneck of solver robustness, which is the mesh quality for 3D icing simulation, is circumvented by using the mixed Cartesian/body-fitted meshing tool (snappyHexMesh) which can generate reasonable mesh on complex icing shapes and still preserve the ice shape. Another important aspect, of a numerical solver, is speed. Here the solver speed at different simulation scenarios are presented.The meshing parameters are carefully adjusted to find a balance between efficiency and accuracy. Although we did not show a pure 2D case in the above sections (just 2.5D with one spanwise layer), it can serve as a good reference for speed estimation. Generally for 2D simulations, the Eulerian method can return a prediction of 360 s of icing time (seven outer icing time steps) around 500 s wall clock on eight Xeon cores. The bottleneck is at the meshing step using the snappyHexMesh. It is because the snappyHexMesh can only generate a 3D mesh. An extrusion process is still required to generate a mesh that has only one layer in the spanwise direction. The Lagrange method may take up to 900 s for the same case. The additional time is spent on particle tracking where load balancing between cores becomes an issue. However, this is not the focus of the current work.For the 2.5D simulations shown in For the M6 wing simulation, the issue of uneven load balancing for the particle tracking is more severe, and the generation of a high-quality true-3D mesh certainly requires more time. It takes about 4–18 h depending on the Eulerian/Lagrangian method selected.This paper introduces a 3D ice simulation code that is developed and implemented in OpenFOAM. Some utilities and solvers already present in OpenFOAM are used and combined to realize the icing simulation. Validations in 2.5D are performed to prove the feasibility and accuracy of the icing simulation ability. It is worth noticing that both the Eulerian method and the Lagrangian method are applicable in the code. For small-sized droplets, the Eulerian method is used. A volumetric fraction is solved with the airflow to replace the droplet tracking process, which is advantageous in solving small-size droplet cases with complex 3D geometries. For SLDs, Lagrangian method is used. Droplets are tracked at individual parcels. The physical characteristics of SLDs can be clearly described, such as splashing or bouncing with the Lagrangian method, which is not investigated much further in this paper. The ice roughness is apparently calculated as a result, which also remains to be further investigated. Finally, a test on the 3D M6 wing is performed to demonstrate the full 3D calculation ability.Much work still needs to be done to develop and improve the code. Besides the code itself, it is worthy of attention that droplet size distribution affects the ice shape greatly, especially the SLD conditions that cover a wide range of the droplet sizes. In this paper, methods of tracking and collecting droplets are separated and the median volume diameter is used as the fixed droplet size instead of the sizes with a distribution. The next step is to make a combination of the Eulerian and the Lagrangian methods to treat the droplets with the given distributed sizes to realize the ability to simulate the ice accretion in the real SLD environments.


Mass loss near the ice-sheet margin is evident from remote sensing as frontal retreat and increases in ice velocities. Velocities in the ice sheet interior are orders of magnitude smaller, making it challenging to detect velocity change. Here, we analyze a 35-year record of remotely sensed velocities, and a 6-year record of repeated GPS observations, at the East Greenland Ice-core Project (EastGRIP), located in the middle of the Northeast-Greenland Ice Stream (NEGIS). We find that the shear margins of NEGIS are accelerating, indicating a widening of the ice stream. We demonstrate that the widening of the ice stream is unlikely to be a response to recent changes at the outlets of NEGIS. Modelling indicates that the observed spatial fingerprint of acceleration is more consistent with a softening of the shear margin, e.g. due to evolving fabric or temperature, than a response to external forcing at the surface or bed. A new study finds that the North East Greenland ice stream is not as stable as previously thought and that this will affect its future evolution. The Northeast Greenland Ice Stream (NEGIS) is a prominent ice-flow feature in Greenland. It is the only ice stream that extends into the interior of the ice sheet, initiating less than 150 km from the ice divide. NEGIS drains ~12% of the ice sheet into the Nioghalvfjerdsfjorden (79 N), Zachariæ, and Storstrømmen glaciers2 . We also use GPS observations from a multi-annual measurement campaign in the vicinity of the EastGRIP deep drilling site , located on NEGIS in the interior of the ice sheet 18.We calculate the acceleration from ice velocity maps spanning the period 1985 to 2018  a thicker ice stream flows faster and but, unlike observations, the velocity anomaly is concentrated in the ice-stream interior; (2) increasing the zone of sliding at the base leads to a spatial map of acceleration that peaks just outside the shear margin; and (3) softening the ice column beneath the shear margin leads to an acceleration that peaks just inside the shear margin.Modeling can help us distinguish between different expected responses to hypothesized forcing scenarios. We run four different experiments with an idealized model of a 2D cross section of an ice stream to investigate the surface velocity response to changes in the boundary conditions and ice-flow parameters (see online methods 4)22 that leads us to conclude that the signal would be greatly dampened at these distances and would likely be undetectable at EastGRIP (see online methods 3). The ice-stream widening might, instead , be a response to an ongoing slow adjustment of the ice-sheet geometry to changes further in the past, such as the last glacial termination, as geometry controls the large scale flow of ice and hydrological drainage23. Finally, the widening could be a response to recent local changes in climate, such as a trend in accumulation. However, this seems unlikely as accumulation trends in this region are small24, and likely have been small for the past several hundred years25. Our modeling finds that the spatial pattern of acceleration is most consistent with a softening of the ice column beneath the shear margin, rather than changes at the surface or the base. The softening could be facilitated via warming or an evolving fabric. This is unlike the basal mechanisms that have been proposed for the reorganization of ice streams in the Siple coast region of West Antarctica16. Given the additional evidence that the interior part of NEGIS is not topographically steered26, we conjecture that the observed widening of NEGIS is indicative of a dynamical instability in the ice stream.The observed widening of the ice stream can either be a response to external forcing or an unforced dynamical instability in the streaming flow. Several kinds of external forcing might explain the observations presented here. E.g., it might be supposed that the interior marginal acceleration of NEGIS is a response to the recent large dynamical changes at the outlets. However, the dynamical response of the outlets and the marginal acceleration are separated by a region with no clear patterns of acceleration (Fig. 2. This could indicate that NEGIS is very susceptible to even slight changes in forcing2. However, the observations could also be partly explained by unforced dynamical instabilities in the ice stream. Regardless of the cause, our study shows that the ice sheet interior is more dynamically variable than hitherto measured, which has deep implications for the future of the ice sheet. This study therefore raises pressing questions: Why is the ice stream accelerating so deep in the interior? Could NEGIS widening be a pre-cursor to a large-scale reorganization of ice flow in North East Greenland similar to changes in the ice streams at Siple Coast, Antarctica28? Our findings highlight the need to understand and carefully separate internal dynamics of ice streams from different forced perturbations and long-term changes in climate forcings. This is crucial if we are to accurately project ice-sheet mass loss in response to recent and future climate change.Further study is needed to understand the mechanism driving the ice-stream widening. A reconstruction of NEGIS frontal fluctuations over the past 45,000 years suggests that relatively large retreats do not have to be associated with large changes in air or ocean temperaturesSupplementary InformationPeer Review File


Arabidopsis arenosa (L.) Lawalrée, alpine populations have evolved independently in different mountain ranges, enabling studying mechanisms of acclimation and adaptation to alpine environments.Success or failure of plants to cope with freezing temperatures can critically influence plant distribution and adaptation to new habitats. Especially in alpine environments, frost is a likely major selective force driving adaptation. In We tested for heritable, parallel differentiation in freezing resistance, cold acclimation potential and ice management strategies using eight alpine and eight foothill populations. Plants from three European mountain ranges  were grown from seeds of tetraploid populations in four common gardens, together with diploid populations from the Tatra Mountains. Freezing resistance was assessed using controlled freezing treatments and measuring effective quantum yield of photosystem II, and ice management strategies by infrared video thermography and cryomicroscopy.The alpine ecotype had a higher cold acclimation potential than the foothill ecotype, whereby this differentiation was more pronounced in tetraploid than diploid populations. However, no ecotypic differentiation was found in one region (Făgăraș), where the ancient lineage had a different evolutionary history. Upon freezing, an ice lens within a lacuna between the palisade and spongy parenchyma tissues was formed by separation of leaf tissues, a mechanism not previously reported for herbaceous species.The dynamic adjustment of freezing resistance to temperature conditions may be particularly important in alpine environments characterized by large temperature fluctuations. Furthermore, the formation of an extracellular ice lens may be a useful strategy to avoid tissue damage during freezing. Arabidopsis arenosa populations, which evolved independently in different mountain ranges, have a higher potential to cold acclimate as compared to foothill populations.Testing for heritable, parallel differentiation in freezing resistance and ice management strategies, this study shows that alpine  Compareet al. i.e. the ability of a genotype/population to produce different phenotypes in response to different environmental conditions. In the long term, specific traits that contribute to higher fitness in a given environment may be favoured and maintained by natural selection, and can become heritably manifest as evolutionary adaptation  Garcke  Lawalrée, an emerging new model plant , Hannah et al. , Zuther et al. , Li et al. , Melcher et al. (Metrosideros polymorpha), Liu et al. (Rhododendron catawbiense) and Armstrong et al. (Arabidopsis kamchatica). A pivotal component of plant freezing resistance is the capability of controlling ice nucleation and ice propagation , Tatra Mountains  and Făgăraș , all showing pronounced, comparable differentiation in environmental conditions between foothill and alpine habitats; see Table et al. , one at 980 m a.s.l. in Aigen im Ennstal, and the other at 2320 m a.s.l. on the northern slope of Mt. Hohenwart  installed in each common garden. Measurements were taken at 6‐min intervals. Rosette leaf temperatures, as typically used for ecophysiological measurements , were recorded using Type‐T copper‐constant thermocouples placed on the lower leaf surfaces of 15 individuals, which were distributed over the entire area  at each of the six sampling dates, corresponding to different acclimation temperatures Table . The numPseudomonas syringae van Hall 1902) to trigger ice nucleation at approximately −2 °C , or within the next 28 h (for leaves transported to the lab from the Styrian common gardens). For each population, seven transparency sheets were prepared, to expose the leaves to six freezing temperature treatments and a control treatment, as described below. On each of the seven transparency sheets, eight to ten randomly selected leaves were taped . When several leaves were collected from the same individual, they were systematically taped to different sheets to be tested at different temperatures. These sheets were immediately covered with a paper towel moistened with ice‐nucleation‐active (INA) bacterial suspension (V/FM)  5 days after the end of the freezing treatment as described in Neuner & Buchner . Soil temperature was precisely controlled during the experiment by using thermocouples placed into the soil: the heating mat was turned on when temperature dropped below 2.6 °C and off when soil temperature reached 5.6 °C, resembling natural conditions during transient frost events, e.g. during nights, when soil remains unfrozen and warmer than the air . Infrared differential thermal analysis (IDTA) was applied, and data were analysed with the ResearchIR software (Flir Systems). By subtracting a reference image captured just before the onset of the freezing event from the subsequent frames during freezing, ice propagation can be monitored at high resolution  from the Botanical Garden Innsbruck  according to Kuprian et al.  and Neunet al. . IDTA alet al. . IDTA exet al.  mounted on the microscope. In addition, cross‐sections of leaves were made with a hand microtome  and observed with a light microscope  controlled by the software cellD (version 3.1).Leaves from plants of all populations  grown in the common garden within the Botanical Garden Innsbruck were fixed in a specimen mount and observed with a reflected‐light microscope  placed inside the cooling compartment of a temperature‐controlled freezer  as described in Stegner et al. , enablinet al. A. arenosa, instead of simple linear regressions. LME allow accounting for two sources of dependence within the data, i.e. population of origin and common garden used as crossed random intercepts. A random factor ‘site’ was used to account for all variation related to the site (transplantation time and location) in all models, except for the comparison of ice nucleation temperatures, which were only measured on one site. One model was used to test for ecotypic differentiation in freezing resistance between foothill and alpine populations, and for parallel evolution of alpine populations in the three mountain ranges. Ecotype, acclimation temperature, mountain range and the interactions between ecotype–acclimation temperature and ecotype–mountain range were regressed as fixed effects on LT50 [LT50 ~ ecotype + Tmean + mountain range + ecotype: Tmean + ecotype: mountain range + (1¦site) + (1¦population)]. Parameters were estimated by optimizing the restricted maximum likelihood criterion. The same model structure, but replacing mountain range by ploidy, was used to test for differences between diploid and tetraploid populations from the Tatra Mountains [LT50 ~ ecotype + Tmean + ploidy + ecotype: Tmean + ecotype: ploidy + (1¦site) + (1¦population)]. Differences in ice nucleation temperatures were tested by using ice nucleation temperature as response and ecotype, mountain range and the interaction of ecotype and mountain range as fixed‐effects [INT ~ ecotype + mountain range + ecotype: mountain range + (1¦population) + (1¦Tmean)]. Determination of ice nucleation was conducted in March and April 2019. To account for differences in temperatures preceding the days of ice nucleation measurements, the 6‐days mean of leaf temperature was calculated and considered as random factor in the model; population of origin was used as second random factor. The assumptions of normal distribution of residuals and random effects and of homogeneity of variances were checked. For each model, marginal (R2LMMm) and conditional R2 values (R2LMMc) were determined using the function ‘r.squaredGLMM’ implemented in the package ‘MuMin’ (Burnham & Anderson Linear mixed‐effects models (LME) implemented in the function ‘lmer’ of the package ‘lmerTest’ , the alpine ecotype had lower LT50 values than the foothill ecotype, but not at the ‘higher’ temperatures (in the range between 12.6 and 17.7 °C). Considering the three mountain ranges separately, this pattern was similar for the Tatra Mountains  than their diploid counterparts after acclimation to falling temperatures , antioxidant levels, cell wall modifications, membrane modifications, hormone balance and regulation of gene expression  of the Arabidopsis arenosa populations of origin.Table S2. Details of common garden locations, transplantation experiments and samplings for freezing resistance assessment.Table S4. Freezing resistance of leaves of alpine and foothill, tetraploid populations of Arabidopsis arenosa.Table S5. Freezing resistance of leaves of diploid and tetraploid populations of Arabidopsis arenosa originating from the Tatra mountains.Click here for additional data file.Table S3. Freezing resistance of leaves of alpine and foothill populations of Arabidopsis arenosa.Click here for additional data file.


The Cortical Bone Trajectory (CBT) technique provides an alternative method for fixation in the lumbar spine in patients with osteoporosis. An accuracy CBT screw placement could improve mechanical stability and reduce complication rates.The purpose of this study is to explore the accuracy of cortical screw placement with the application of implanted spinous process clip (SPC) guide.Four lumbar specimens with T12-S1 were used to access the accuracy of the cortical screw. The SPC-guided planning screws were compared to the actual inserted screws by superimposing the vertebrae and screws preoperative and postoperative CT scans. According to preoperative planning, the SPC guide was adjusted to the appropriate posture to allow the K-wire drilling along the planned trajectory. Pre and postoperative 3D-CT reconstructions was used to evaluate the screw accuracy according to Gertzbein and Robbins classification. Intraclass correlation coefficients (ICCs) and Bland–Altman plots were used to examine SPC-guided agreements for CBT screw placement.A total of 48 screws were documented in the study. Clinically acceptable trajectory (grades A and B) was accessed in 100% of 48 screws in the planning screws group, and 93.8% of 48 screws in the inserted screws group (p = 0.242). The incidence of proximal facet joint violation (FJV) in the planning screws group (2.1%) was comparable to the inserted screws group (6.3%) (p = 0.617). The lateral angle and cranial angle of the planned screws (9.2 ± 1.8° and 22.8 ± 5.6°) were similar to inserted screws . Reliability evaluated by intraclass correlation coefficients and Bland–Altman showed good consistency in cranial angle and excellent results in lateral angle and distance of screw tip.Compared with preoperative planning screws and the actually inserted screws, the SPC guide could achieve reliable execution for cortical screw placement. A cortical bone trajectory (CBT) is a novel technique providing an alternative approach for pedicle screw placement in lumbar spinal surgery . The entPrevious studies have reported the accuracy of cortical screw placement with the assistance of 3D printing guides, 3D fluoroscopy-assisted navigation technology, and robot-assisted screw implantation . AlthougThis study has provided the initial experience of the application of spinous process clamp (SPC) guided cortical screw placement and evaluated the accuracy of the device. The SPC guide was a device that anchored on the spinous process of the surgical segment vertebral body through a special tooth-like clamp, providing cortical trajectories for patients requiring spinal fusion between L1 and S1. According to the preoperative 3D-CT reconstruction, planning adjusts the position and posture of the SPC guide during the procedure to make sure that the screw was inserted precisely along the cortical trajectory.Four formalin-treated cadaveric lumbar specimens with T12–S1 segments were obtained from the China Capital Medical University for this study. All the cadaver specimens without congenital spinal deformity, pars defect, metastatic spinal lesions, trauma, infection, and history of spinal surgeries were enrolled. Paravertebral soft tissue on the lamina, pars interarticularis, and facet joint were fully stripped, but the interspinous and supraspinous ligaments were preserved. All processed specimens were temporarily stored in a refrigerator at − 20 °C until they were warmed to room temperature for further using. A total of 48 screws were placed after the cortical trajectory planning at L1 to S1. All screw placement were performed by two spine surgeons at our center with extensive experience in CBT screw placement. Screw parameters include lateral angle (LA), cranial angle (CA) and distance between screw tip (DBST) were measured in preoperative planning and actual inserted lumbar specimens. LA was defined as the angle between the symmetry axis of the vertebral body and the axis of the cortical screws. CA was defined as the angle between the cephalad endplate line and the axis of the cortical screws, DBST was the distance between two screw tips measured from 3D CT reconstructions.® . The three-dimensional cortical screw trajectory planning including the starting point, CA, LA, screw length, screw diameter, and the length between the spinous process to knob was achieved by Solidworks®   . 3D lumbar models were reconstructed by MimicsShown in the Fig. As shown in Fig. 3D reconstructions of vertebrae and insert screws were established from postoperative CT scans Fig. . PreoperContinuous data are listed as means ± SD for normal distribution, while non-continuous data were presented as numbers or ratios. The reliability of the parameters documented in two groups with SPC guide were determined by intraclass correlation coefficients (ICCs). Reliabilities > 0.75 were considered as excellent, 0.40–0.75 as fair to good, < 0.40 were characterized as poor. All statistical analyzes were calculated by SPSS Statistics 20 . Bias of the data conforming to normal distribution were analyzed by Bland–Altman analysis to evaluate the agreement by GraphPad Prism 8 .A total of 48 CBT screws were implanted into four lumbar specimens in this study, each lumbar spine specimen was implanted 12 CBT screws in the L1-S1 segment. The planning accuracy of CBT screws were evaluated by the software before the surgery, after the CBT screws were implanted according to preoperative planning, the accuracy of inserted screws was evaluated from 3D-CT reconstructions. Meanwhile, the patient demographics and parameters were shown in Table The CBT screw accuracy grades was shown in Table Due to the preoperative modeling to visualized planning screw placement, the screw deviation only with grades A and B. Table The screw parameters were measured in planning screws and inserted screws as shown in Table For measurements of CA, the consistency was good for ICC at 0.746 (95%CI 0.588–0.849). For LA and DBST, excellent reliabilities were demonstrated   screw technique was described to enhance the fixation strength by maximizing the screw thread contact with the cortical bone . The rigHowever, there were some disadvantages of those novel screw insertion technology. The paraspinal muscles, ligaments, and facet joint capsule needed to remove for the 3D printed guide template close contact with the spinous process and lamina [This study initially described the accuracy of a cortical screw inserting utilized the SPC guide. A total of four lumbar spine specimens with L1-S1 vertebrae contained twelve screws implanted in each specimen, for a total of 48 screws. Our results demonstrated that application of the SPC guide during cortical screw placement could provide reliable execution of preoperative screw planning and accuracy and safety were improved significantly. The clinically acceptable screw placement (corresponding to grade A and grade B screws) for the planning screw (93.8%) was comparable to the accuracy of the inserted screw (83.3%) (p = 0.242). The incidence of proximal facet joint violation (FJV) for planning screws (1.2%) was comparable with inserted screws (6.3%) (p = 0.617), which indicated that the SPC guide was fixed on the spinous process firmly during the screw implant progress.®  for 3D modeling of the spine, planning the SPC guide to implant the screw under the condition of the whole spine visualization can avoid the screw thread penetrating the outer cortex of the pedicle vertebral body as precise as possible. However, in this study, there were three planning screws record as grade B, the possible explanation was that the planned K-wire inserted the vertebrae through the SPC guide was much thinner than the screw model. Despite the pedicle along the cortical trajectory could insert a planned K-wire, it was still too narrow to insert a CBT screw, and the plan was used to minimize the perforated incidence. The explanation of inserted screw placement as grade B or C was that the putation disadvantage of a K-wire-based guide through SPC guide has a high risk for bowing because its diameter was too thin to keep straight.The deviation of the planning screw and the inserted screw were measured from the superimposed 3D reconstruction of the screw and vertebrae pre- and postoperative. The accuracy of the CBT screw placement was graded according to the modified method of Gertzbein and Robbins, . Since the preoperative screw planning was carried out in the MimicsThe direction of the cortical screw perforation including caudal pedicle, medial pedicle anterior and lateral walls of the vertebral body, and cephalad endplate results in nerve root irritation, spinal cord injuries, disc degeneration, vascular, and biomechanical defect –13. In tIn the sagittal plane, the caudal deviation can jeopardize neural tissue, cranial deviation causes proximal facet joint violation (FJV), and cephalad endplate led to disc degeneration. The present study demonstrated that the most common deviation was cephalad endplate followed by caudal perforation. The anchor site of the SPC guider with clamp was planned before surgery and pinched into the spinous process, the cranial angle of the column of the guide was adjusted based on preoperative planning, a K-wire was stabbed into the spinous process through the column and checked with lateral imaging at last. During this process, there will be an acceptable slight displacement of the clip and the spinous process, and the angle of the caudal inclination of the post will increase due to the influence of gravity, which can cause cephalad endplate perforation. With the increased caudal inclination of the SPC guide, the guided screw is more likely to involve the proximal facet joint.Parameters of any deviation of inserted screws tips from planned screws were measured through the preoperative and postoperative 3D-CT reconstructed vertebrae and screws superimposed. Our results demonstrated no statistically significant difference between the distance of planning screws tip and inserted screws tip. The tips of the screws were selected as the cortical screw deviation parameter which was measured with 3D models reconstructed from CT scan. Due to the screw entry point is the anchor for screwing the screw, the position is constant with a slight deviation. The deviation of the screw direction during the insertion process will eventually be enlarged at the screw tip, so the screw tip is the best indicator for measuring the screw deviation.The present study has some limitations. First, the SPC guide was adjusted manually based on the preoperative cortical screw parameter, which enlarged the deviation during the insertion of screws. Second, the small sample size of this study limits the evaluation of screw accuracy, and the conclusions drawn lack credibility and representativeness. Third, further clinical studies are needed to evaluate the safety, efficacy of screw insertion with the SPC guide. Finally, when forced to adjust the SPC guide may cause spinous process fractures.In conclusion, this cadaveric study shows that the influence of gravity on the SPC guide can lead to an increase in the cranial angle of the screw. Although it may increase the incidence of penetration of the cranial wall of the pedicle and the cephalad endplate of the vertebral body, it will reduce the penetration of the caudal pedicle wall, which enable lead to serious neurological complications. Restricted by the spinous process, the screw planned by the SPC navigator is lateral and has a larger abduction angle. Although it will increase the damage of the pedicle or the lateral wall of the vertebral and also reduce the damage of the medial wall that leads to nerve root damage. The SPC guide screws placement accurately and reduces severe deviations in important directions. More clinical results are needed to confirm the imaging and clinical effects in the future.