Droplets or puddles tend to freeze from the propagation of a single freeze front. In contrast, videographers have shown that as soap bubbles freeze, a plethora of growing ice crystals can swirl around in a beautiful effect visually reminiscent of a snow globe. However, the underlying physics of how bubbles freeze has not been studied. Here, we characterize the physics of soap bubbles freezing on an icy substrate and reveal two distinct modes of freezing. The first mode, occurring for isothermally supercooled bubbles, generates a strong Marangoni flow that entrains ice crystals to produce the aforementioned snow globe effect. The second mode occurs when using a cold stage in a warm ambient, resulting in a bottom-up freeze front that eventually halts due to poor conduction along the bubble. Blending experiments, scaling analysis, and numerical methods, the dynamics of the freeze fronts and Marangoni flows are characterized. While the physics of freezing water droplets are known, it is less known how bubbles freeze. The authors investigate the physics of freezing soap bubbles and identify two distinct freezing modes, depending on whether the surroundings are warmer or colder than the melting temperature. Building on this foundation, in modern times, the behavior of bubbles from their birth6, wetting7, drainage and evaporation9, to their fatal bursting11 has been comprehensively studied. This has culminated in the practical use of bubbles for a myriad of applications such as energy harvesting12, drug delivery13, and cleaning devices16.Soap bubbles and films have been a source of intrigue for millennia. Their influence can be traced in a historical arc that includes Babylonian divination rituals (lecanomancy) to Impressionist paintings to the works of physicists such as Newton, Plateau, and de Gennes17. In the field of visual arts, on the other hand, there is an emerging trend of photographers capturing beautiful videos of the complex freezing dynamics of bubbles deposited on snow . These dynamics are nontrivial owing to the unique geometry of a bubble: unlike droplets, puddles, or surface-bound liquid films, bubbles do not have a thermally conductive bulk volume. Therefore, the extensive studies of how droplets25 or films28 freeze cannot capture the physics of bubble freezing.Despite this prolonged attention lavished on bubbles, there exists only a brief scientific report of freezing. In 1949, Shaefer observed bubbles freezing atop Mt. Washington and commented on the number and shape of ice crystals contained thereinInspired by these informal observations of freezing bubbles, here we characterize the heat transfer phenomena governing the dynamics of freezing bubbles over a wide range of conditions. Two different types of freezing dynamics were observed, depending on the experimental conditions. For bubbles freezing in an isothermal environment, the bottom-up freeze front produces a Marangoni flow that detaches ice crystals, resulting in accelerated freezing from multiple fronts growing in tandem. Conversely, when bubbles are deposited on a chilled icy substrate in room-temperature conditions, the bottom-up freeze front comes to a halt midway up the bubble due to poor conduction.T∞) and substrate temperature (Tw) far beneath the melting point (Tm): T∞ ≈ Tw = −18 ± 2 °C < Tm 29. When a calm bubble is punctured, a hole opens and grows due to the unstable surface tension forces at its rim. Using the Dupré–Taylor–Culick law, the initial film thickness of a liquid bubble (e0) can be determined from the hole-opening velocity by balancing surface tension and inertia31:γ = 24.2 mN m−1 is the solution’s surface tension measured by using the pendant drop method and waiting until the surfactant had reached a steady-state packing density at the free interface  as that of pure water: ρ ≈ 1000 kg m−3. From Eq. e0 ≈ 4.7 μm for Ω = 5 μL, e0 ≈ 2.7 μm for Ω = 500 μL, and e0 ≈ 1.7 μm for Ω = 10 mL.For bubbles in both sets of experiments, we used a glycerol–water soap solution exhibiting a freezing point of v ≈ 10 mm s−1 emanating from the early freeze front , buoyant flow due to thermal effects, or a thermal Marangoni flow induced by the latent heat of fusion. The first two flow mechanisms do not require a temperature gradient and can therefore be evaluated using bubbles deposited on a dry substrate at room-temperature conditions Fig. . The dismentclass2pt{minim33, is purely geometric and owes its origin to the liquid meniscus at the foot of the bubble. The Laplace pressure difference between the bubble and the meniscus generates plumes that are long-lived34. This agrees with our control case of room-temperature bubbles, where the plumes were maintained for most of the lifetime of the bubble  = ρVe0/η, where η ≈ 2 × 10−3 Pa s is the viscosity of water36 at −6.5 °C. For typical values of V~10 mm s−1, as measured by observing the initial speed of a rising plume .A previous work has shown that buoyant thermal plumes can be generated in vertical soap films, where the plumes were primarily inertialume Fig. , we get γ/δ, where δ is the length scale of the temperature gradient driving the flow. This must be balanced by viscous stress, ηVM/(b + e0/2), where VM is the Marangoni velocity, b is the slip length of the Poiseuille flow along the bubble’s film ~e0, resulting in a simplified viscous stress of δ:This leaves us with the final possibility of a Marangoni flow induced by the latent heat released from freezing. We will refer to this process as “Marangoni freezing.” The freezing-induced heating engenders a gradient in surface tension, Δγ ≈ 2 mN m−1 for ΔT = Tm − Tl ≈ 13.5 °C corresponding to our degree of supercooling  are nontrivial and beyond the scope of this research, as has been noted before in the phenomenon of Marangoni bursting38.Note that Δing Fig. 37. When Tw < Tm, and the condition for vertically upward Marangoni flow: dT/dz < 0, such that dγ/dz > 037. For the Marangoni flow to dominate, it must be at least as fast as the rate of thermal diffusion, VT~αl/δ, where αl = 0.13 × 10−6 m2 s−1 is the thermal diffusivity of the liquid solution. This first flow criterion is stated in terms of the ratio of the Marangoni velocity, VM~Δγe0/ηδ , where no freezing/nucleation events were observed even after 30 min  with the pull-off force required to crack an ice dendrite free of its icy substrate (Fcrack). For a dendritic contact area of πl2, the pull-off force can be determined using the Griffith condition for crack initiation39wad is the work of adhesion and Ei = 8.7 × 109 Pa and νi = 0.31, are the Young’s modulus and Poisson’s ratio corresponding to ice, respectively40. The work of adhesion can be quantified as wad ≈ γi,l where γi,l is the interfacial energy of ice with respect to liquid determined from Young’s relation, γi,l = γi,v − γcosθ. Here, γi,v ≈ 0.1 J m−2 is the interfacial energy of ice with respect to vapor41, γ ≈ 0.02 J m−2  supercooled liquid with temperature Tl < Tm is exposed to a temperature Tc at its boundary (y = 0) at time zero. The tip velocity is then given byλ ≈ 0.03 was obtained from the root of a transcendental equation )/L, L = 334 × 103 J kg−1 is the latent heat of fusion. The tip diameter was crudely measured as 2Ri ≈ 1.28 μm . This, however, has no effect on the initial stages of freezing, as the latent heat released in these experiments to create a local region of dT/dz < 0. However, the negative temperature gradient is only a transient effect, such that the plumes would die out within about 5 s. Perhaps, due to the transient nature of Marangoni freezing for the case of a room-temperature ambient, the “Snow-Globe Effect” was not observed. For the larger Ω = 500-μL bubble, it is also possible that nanoscale ice particles could be entrained in the Marangoni flow, but quickly melted by the warm top of the bubble (Tt > Tm) before growing to a microscale size.Marangoni freezing: Analogous to the walk-in freezer experiments, even in room-temperature experiments, we see that mere milliseconds after deposition of bubbles on a chilled icy substrate, there is a burst of Marangoni plumes moving upward from the contact line. The difference between the two experiments is the initial imposed positive temperature gradient across the bubble in room-temperature experiments , where ci = 2.027 × 103 J kg−1 K−1 is the specific heat capacity of ice and ΔTi~10 K is a typical temperature difference. The diffusion timescale is about an order of magnitude smaller than the freezing timescale, τf~10 τD, which allows us to assume a quasi-steady temperature profile: ∇2T = 0. This remains true for water, as its heat capacity is about two times higher than that of ice.The observed trend of incomplete freeze fronts can be rationalized by the poor thermal conductance of the long and slender soap film. Conservation of energy within the bubble’s film can be expressed as dζ ≈ 3 cm was obtained, in agreement with experimental measurements, using a thermocouple and translation stage , while heat was conducted into the freeze front for the 500-μL bubbles  = Rψ(t), is captured by Eq. β1 = 1.8 and β2 = 30. Therefore, all of the data collapse onto a universal nondimensional curve, as shown in Fig. Canceling like terms and using a dimensionless time ψ/dt* → 0 at a critical angular coordinate ψ→ψf (S→Sf). The energy equation is then simplified to ψf can be found asThe freeze front stops propagating as dhf = 2Rsin((ψf−ψ0)/2) , which is close to ki/kl = 3.93 corresponding to that of pure water. This discrepancy is due to the existence of glycerol, soap solution, and low temperatures. Three distinct regimes of freezing are possible: the nonfrozen regime, where Tw ≥ Tm and ψf → ψ0, the completely frozen regime, where Tt ≤ Tm such that ψf → π, and the partially frozen regime, where Tt > Tm > Tw and ψ0 < ψf < π /(ρgb2)~102−103 s for R~1–10 mm8.Marginal regeneration: Once a bubble reached its partially frozen equilibrium, initially there was no appreciable flow in the upper liquid portion of the bubble. After about 100 s, there was a sudden reappearance of plumes within the liquid dome /2h is the liquid’s radius of curvature, a is the fixed contact radius of the liquid/ice interface, and h is the height of the liquid dome (3a2 + h2) is the volume of the liquid spherical cap and p is the pore radius. Taking the derivative of the volume with respect to h, the change in height of the liquid dome with respect to time isρair = P/(RsTair), where P is the absolute pressure, Rs = 287.058 J kg−1 K−1 is the specific gas constant, and Tair is the average temperature inside the bubble, which was calculated from the computational results . In this region, the “Snow-Globe Effect” is observed when dT/dz ≤ 0, or in other words, Tt ≤ Tw . This condition is satisfied inside the walk-in freezer, where the substrate temperature was equal to the temperature at the top of the bubble. Note that all types of freezing tended to produce some degree of flow due to Marangoni freezing, even though the “Snow-Globe Effect” only occurred for the isothermal case.Figure 8. During the short time where the bubble was intact, the bottom-up freezing dynamics were equivalent to that with the dish soap. For the isothermal conditions, Ω = 500-μL bubbles containing SDS burst within Finally, control tests were performed with a 1% concentration of a pure surfactant, sodium dodecyl sulfate (SDS), rather than 1% dish soap. Bubbles with SDS bursted within In conclusion, the freezing dynamics of soap bubbles are multifaceted and fundamentally distinct from the classically studied scenario of freezing bulk volumes of liquid. Under chilled and isothermal conditions, hundreds of ice particles are detached from the freeze front and swirl around the bubble in a “Snow-Globe Effect.” This beautiful dance was caused by the local input of latent heat at the freeze front, causing a strong Marangoni flow capable of breaking off small ice crystals. Isothermal bubbles therefore freeze very efficiently due to hundreds of fronts growing in tandem and interlocking together. When a bubble is frozen on a cold stage in a room-temperature environment, the freeze front slowly propagates upward and comes to a complete stop at a critical height. The freeze-front dynamics were found to be captured by a Stefan problem governed by a balance between latent heat and conduction across the frozen and unfrozen portions of the bubble. After the halting of the freeze front, the partially frozen bubble remains in equilibrium for many minutes, followed by the deflation and collapse of the liquid dome, due to its Laplace pressure forcing air through small pores in the ice. These findings show that the dynamics of freezing liquid is highly dependent on its geometric conditions, and that a rich variety of multiphase phenomena occur when a liquid volume is neither continuous nor surface-bound.®, Ultra AntiBacterial Dish Liquid). The dish soap consists of 98% inert ingredients , and 2% active ingredient (L-lactic acid). For the control experiments, instead of dish soap, sodium dodecyl sulfate  was used. After stirring the mixture together, it was allowed to sit overnight before running any experiments. Neglecting the effects of the soap, the freezing temperature of an 80–20% water/glycerol mixture was previously reported29 to be Tm ≈ −6.5 °C.The soap bubbles were generated using a solution consisting of 79% (by volume) distilled water, 20% glycerol , and ≈1% dish soap  with a macro lens (Canon MP-E 65 mm f/2.8 1–5×). After freezing, the pores and/or bubbles trapped within the ice were characterized using a top-down optical microscope (Nikon 150LV) with a long working-distance lens .For the room-temperature experiments, bubbles were deposited on a frosted Peltier stage  using a syringe pump . A needle with inner and outer diameters of 0.7 mm and 2.1 mm, respectively, was used. Experiments were recorded using a DSLR camera (Canon®) when transporting to the walk-in freezer . An ice disk was allowed to sit within the walk-in freezer for at least an hour before beginning experiments, to ensure that its temperature was that of the air. For some control experiments, the bubbles were deposited on a dry silicon substrate rather than an ice disk. Experiments were recorded using a high-speed camera . The relative humidity and air temperature of the walk-in freezer were measured by a hygrometer . Thermal imaging experiments were conducted using an IR camera (FLIR SC655).For the isothermal experiments, a polystyrene Petri dish  was filled with distilled water and left in a freezer  with a temperature of −20 °C overnight. Ice disks were kept within a cooler , while a square LED was used for a backlight . When imaging a frozen bubble with the top-down microscope, LED lighting was used to minimize heating effects . For the isothermal experiments, a round LED light  was placed underneath the ice disk with a horizontal orientation to illuminate the bubble  to track three points  of the freezing bubbles. The coordinate system was placed such that the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


IgE-targeted medicines such as FDA approved drug Xolair  represent a promising avenue for treating IgE-mediated allergic diseases given the pernicious role of IgE in disease progression. Additionally, targeted therapy for IgE-mediated allergic diseases may be advanced through cellular treatments, including the modification of effector cells.Allergic diseases are inflammatory disorders that involve many types of cells and factors, including allergens, immunoglobulin (Ig)E, mast cells, basophils, cytokines and soluble mediators. Among them, IgE plays a vital role in the development of acute allergic reactions and chronic inflammatory allergic diseases, making its control particularly important in the treatment of IgE-mediated allergic diseases. This review provides an overview of the current state of IgE targeted therapy development, focusing on three areas of translational research: IgE neutralization in blood; IgE-effector cell elimination; and IgE The incidence of allergic diseases has been rising with increasing industrialization and the accompanying changes to the environment and people’s lifestyles. According to a 30-nation/region epidemiological investigation conducted by the World Allergy Organization (WAO) Specialty and Training Council, approximately 250 million (22%) of the 1.2 billion people in those regions suffered from allergic diseases, such as allergic rhinitis, allergic asthma, allergic conjunctivitis, eczema, food allergies and drug allergies etc. .The mechanism of allergic diseases is complex. In clinical practice, allergic diseases could be divided into two categories: IgE mediated and non-IgE mediated. Allergic disorders are characterized by inflammatory responses involving many types of cells, including mast cells and basophils, and various biological molecules, including cytokines (e.g. interleukin 4) and soluble mediators (e.g. histamine) . IgE, anIgE shares the same basic molecular structure of other Igs, with two identical heavy chains and two identical light chains. However, the heavy ε-chain of IgE contains one more domain than the heavy γ-chain of IgG. The Cε3 and Cε4 domains of IgE are sequence homologous and structurally similar to the Cγ2 and Cγ3 domains of IgG; two Cε2 domains, which are the most obvious distinguishing feature of IgE, are present in place of the flexible hinge region found in IgG –10. The 2+, thereby triggering an allergic reaction [IgE is a glycoprotein with a molecular mass of 190 KD and a sedimentation coefficient of 8S . It is nreaction .+ B cells start to secrete free IgE into the blood where it can bind the high-affinity IgE receptor FcεR I on mast cells or basophils [IgE plays a key role in mediating the initiation and development of allergic diseases. The IgE molecule performs its biological function by binding its receptors on target cells, activating the induction immunomodulatory and protecting against parasitic worms (helminths) and the expulsion of environmental substances that include toxins, venoms, irritants and xenobiotics . When thasophils . This reasophils . Apart fasophils . Surfaceasophils . Anti-Igasophils , 21. Witasophils .There is great interest in the development of new drugs or methods that would alleviate allergic diseases by affecting molecular IgE activities in a manner that is safe, effective, and convenient Table .Table 1TIgE is an important target for allergic disease therapy. The main method of IgE neutralization being pursued involves achieving specific binding and neutralization of free IgE in serum to prevent it from binding receptors on target cells, thereby inhibiting allergen-induced early/late allergic reactions Fig. .Fig. 1SImmunoadsorption (IA), also called immune apheresis, has been adopted as an effective treatment for autoantibody-mediated diseases . IA util®, is a recombinant humanized monoclonal antibody against IgE [Omalizumab, developed by Novartisinst IgE . It bindinst IgE . Becauseinst IgE . In addiinst IgE .However, omalizumab is associated with safety concerns. Because it cannot reduce IgE levels quickly, it requires a long , continuous treatment cycle . The US In addition, CMAB007, a biosimilar of omalizumab, was developed by National Engineering Research Center of Antibody Medicine of China. It has the same amino acid sequence as omalizumab and have been finished phase III clinical trial in China approval by local departments . Now, th®, is a humanized IgG1 monoclonal antibodytargeting the Cε3 region of IgE [Ligelizumab, also developed by Novartisn of IgE . Like omPhase II clinical trials investigating the pharmacokinetics, pharmacodynamics, and safety of ligelizumab showed that it can decrease IgE levels more effectively than omalizumab via inhibition of IgE-FcεRI binding and that it can produce better outcomes, as indicated by skin prick allergen test responses , 39. HenMEDI4212, a humanized IgG1λ monoclonal antibody generated by phage display technology, neutralizes free IgE by binding selectively to the Cε3 and Cε4 domains of IgE . The affA phase I clinical trial (NCT01544348) showed that MEDI4212 is more effective than omalizumab in reducing serum IgE levels inpatients with IgE levels ≥ 30 IU/mL and that MEDI4212 treatment reduces FcεRI expression on dendritic cells and basophils . However+ B cell elimination.The MEDI4121 may be mutagenized to improve the drug’s pharmacokinetic properties. A variant of MEDI4121 in which the Fc fragment was altered to enhance affinity for FcγRIIIa, which down-regulates IgE expression by B cells before they have differentiated into IgE secreting plasma cells . Therefo−10 M) [+ B cells) as well as free IgE in vivo [+ effector cells or trigger basophil/mast cell degranulation. Recombinant ScFv can also be used to probe IgE activities under both healthy and disease conditions, making it a helpful drug development tool.Recombinant ScFv is produced by cDNA encoding the heavy and light Ig chains. Biosensor-based studies have demonstrated that recombinant ScFv binds human IgE rapidly and efficiently  . It can  in vivo . In vitrImmune effector cells, including mast cells, basophils, eosinophils, which are critical for the elimination of foreign substances and antigens, are the main sources pro-inflammatory factors. In particular, mast cells are the key cells that induce allergic asthma, and the number of mast cells in asthmatic patients is elevated significantly , 45. AftTripterygium wilfordii (Thunder god vine) that can induce T cells apoptosis [Celastrol is bioactive compound extracted from poptosis . Thus, tpoptosis . Treatmepoptosis . The abiAnti-FcεRIα Fab-conjugated polymeric micelles have been shown to reduce allergic reactions more efficiently than omalizumab . The fol+CD25+ Foxp3+regulatory T cells, but is upregulated only in conventional T cellsupon activation. CTLA4 is homologous to the T cell co-stimulatory proteinCD28, and both molecules bind CD80(B7-1) and CD86(B7-2) on antigen-presenting cells [CTLA4  is a protein receptor that, functioning as an immune checkpoint, down-regulates immune responses. It is constitutively expressed in CD4ng cells . It bindng cells . CTLA4 tng cells .Researchers have constructed a fusion protein containing the CD80/CD86-binding domain of CTLA-4 and the Fcε receptor-binding domain of the IgE H chain . This re+ B cells are critical for controlling IgE production. Both transient IgE secreted by plasma blasts in blood and long-living IgE secreted by plasma cells in bone marrow are influenced by IgE+ B cells [IgE B cells .Membrane-bound IgE on the surface of B lymphocytes is of great importance for IgE production. It has an extra 52-amino acid-long CεmX-containing fragment between the CH4 domain of IgE and its B-cell membrane-anchoring segment . CεmX is®, is a new artificial monoclonal antibody that targets CεmX on IgE+ B cells. It produces crosslinking of membrane-bound IgE antigen receptors on B cells, which induces IgE+ B cell apoptosis, thereby reducing free IgE levels and inhibiting the generation of IgE+ B cells [Quilizumab, developed by Genentech B cells , 57. Bec B cells .A phase II clinical trial showed that quilizumab is an effective candidate for treating allergic diseases safely and with high specificity . Quilizu+ B cells, including plasma blasts, plasma cells, and IgE+ memory B cells [+ B cells specifically sought to modify T-cell receptors in combination with anti-IgE monoclonal antibody activity [+ B cells, at least in vitro, without causing degranulation of mast cells or release of free IgE. Bsc-IgE/CD3 is an antibody that could eliminate both IgE+ B cells and free IgE in serum [There are several types of IgE B cells . The earactivity . The bscin serum . Therefo+ B cells and reduce free and total IgE levels without affecting the antigen isotypes of other B cells [+ B cell differentiation, thereby reducing the number of IgE secreting plasma cells [FcγRIIβ is involved in B-cell homeostasis and FcγRIIβ abnormalities lead to autoimmune diseases . A novel B cells . Becausema cells . This rema cells .+ B cells are also in development. Apart from targeting medicines, cellular treatments are also being developed. For example, T cells may be modified to bind anti-IgE T cell receptors, thereby reducing the number of IgE+ cells [As so far, Omalizumab is the only FDA-approved recombinant humanized monoclonal antibody that neutralizes IgE to treat allergic diseases . Its useE+ cells .®) was approved by the FDA in April 2017 for the treatment of adult patients with moderate-to-severe atopic dermatitis [It is a great progress that another new drug dupilumab (Dupixent rmatitis . Dupilumrmatitis .IgE-targeting is highly promising for the treatment of allergic diseases. Cellular treatments, including selective modification of effector cells, represent an innovative technology for targeted therapy of allergic diseases . Notwith+ B cell reduction. More IgE-targeted medicines such as FDA approved drug Omalizumab are expected for treating IgE-mediated allergic diseases in the future.IgE is an important therapeutic target in IgE-mediated allergic diseases. Novel IgE targeted therapies are currently being developed, which focus on IgE neutralization in blood, IgE-effector cell elimination and IgE


IgE-mediated allergic disease represents an increasing health problem. Although numerous studies have investigated IgE sequences in allergic patients, little information is available on the healthy IgE repertoire. IgM, IgG, IgA, and IgE transcripts from peripheral blood B cells of five healthy, non-atopic individuals were amplified by unbiased, template-switching, isotype-specific PCR. Complete VDJ regions were sequenced to near-exhaustion on the PacBio platform. Sequences were analyzed for clonal relationships, degree of somatic hypermutation, IGHV gene usage, evidence of antigenic selection, and N-linked glycosylation motifs. IgE repertoires appeared to be highly oligoclonal with preferential usage of certain IGHV genes compared to the other isotypes. IgE sequences carried more somatic mutations than IgM, yet fewer than IgG and IgA. Many IgE sequences contained N-linked glycosylation motifs. IgE sequences had no clonal relationship with the other isotypes. The IgE repertoire in healthy individuals is derived from relatively few clonal expansions without apparent relations to immune reactions that give rise to IgG or IgA. The mutational burden of normal IgE suggests an origin through direct class-switching from the IgM repertoire with little evidence of antigenic drive, and hence presumably low affinity for specific antigens. These findings are compatible with a primary function of the healthy IgE repertoire to occupy Fcε receptors for competitive protection against mast cell degranulation induced by allergen-specific, high-affinity IgE. This background knowledge may help to elucidate pathogenic mechanisms in allergic disease and to design improved desensitization strategies. Type I hypersensitivity is an immune response triggered by allergen-specific IgE. Binding of allergen-bound IgE to Fcε receptors on mast cells leads to prompt degranulation, which provokes a range of clinical symptoms including atopic dermatitis, asthma, allergic rhinoconjunctivitis, urticarial, and anaphylaxis , 2. DespTwo distinct pathways to generate IgE have been identified in mouse models. In the direct pathway, functional B-cell receptor (BCR) genes undergo direct class-switch recombination (CSR) from IgM to IgE isotype and yield predominantly IgE with low affinity to antigen. In the indirect pathway, high-affinity IgE results from secondary CSR in B cells that express IgG or potentially IgA , 9. AlonSince low and high affinity IgE compete for occupation of the Fcε receptor, higher concentrations of low-affinity IgE than high-affinity IgE can theoretically provide protection against anaphylaxis . In non-This study aimed to provide in-depth characterization of the IgE repertoire in healthy, non-allergic individuals as an essential reference for comparative studies in allergic and desensitized individuals. We determined the peripheral blood IgE BCR repertoire of five non-allergic donors to near completion by unbiased, full-length massive parallel sequencing. IgE BCR repertoires were compared to IgM, IgG, and IgA repertoires to support the hypothesis that the direct pathway of IgE generation would be dominant in non-allergic human individuals.Cryopreserved aliquots of Ficoll-separated mononuclear cells (PBMC) from five healthy, asymptomatic stem cell donors were obtained from the biobank of the Leiden University Medical Center biobank in accordance with local guidelines. Absence of atopic constitution was confirmed by measurement of total IgM, IgG, IgA, and IgE immunoglobulin levels in time-matched serum samples.5 thawed PBMC by flow cytometry. To avoid artifacts from binding of IgE to Fcε receptor-expressing cells  of the IgE-expressing multiple myeloma cell line U266  in a background of healthy donor PBMCs. For IgE repertoires, cDNA was first amplified for 15 cycles with the ε.pcr1 primer. First-round IgE amplicons were purified by silica spin columns  and re-amplified for 15 cycles of semi-nested PCR using the ε.pcr2 primer. Libraries were barcoded at the 5′ terminus to identify the donor and at the 3′ terminus to identify individual aliquots. Pooled libraries were amplified as single molecules in rolling circles on a total of five SMRT cells on the RSII system . IgE sequences were sequenced to high depth to achieve near-complete representation of all IgE+ B cells present in the sample.Messenger RNA isolation and cDNA synthesis were performed separately for each aliquot with addition of the ε.rt primer to the ARTISAN cDNA synthesis mix . IgM, IgOutput sequence files were filtered with SMRT portal software for a minimum of eight sequencing passes. All sequences were annotated by IMGT HighV-QUEST . For stat-test.IGHV gene usage was compared between isotypes by Fisher's exact test and corrected for multiple testing by Bonferroni correction. Cumulative differences in IGHV gene usage were calculated by determining the absolute differences in fractional IGHV usage between two isotypes for every IGHV gene and adding these to a cumulative difference with a theoretical maximum of 200. BCR mutational status and CDR3 length were compared between isotypes by unpaired N-linked glycosylation motifs were identified as Asn-X-Ser/Thr motifs (where X may be any amino acid except proline) and their abundance, location, and the mutational status of the corresponding sequence were compared between isotypes using unpaired t and Fisher's exact tests.Clonal B-cell expansions were defined as the presence of BCR sequences with identical V, D and J gene usage, identical CDR3 length and ≥95% nucleotide overlap in CDR3 in either multiple aliquots or in more than one isotype of any individual donor. Intraclonal sequence variation was defined as the distance from the clonal consensus and determined for the largest 10 IgE clones, as well as the largest 5 IgM, IgG, and IgA clones.in silico simulation to estimate the fraction of observed unique sequences sampled out of a large pool of sequences. A unique numerical identifier was assigned to each unique observed BCR sequence for each donor, and 5 × 105 copies of every identifier were pooled in the simulation. Consequently, random sequences were sampled from the pool up to the number of sequences obtained from massive parallel sequencing, and the number of unique identifiers was counted. This simulation was performed 100 times per donor, and the median number of unique samples identifiers per donor was calculated.To assess how many cells from the sample were represented in the VDJ sequence libraries, we performed an According to applicable stem cell donor regulations, all donors had no atopic constitution, denied any allergic symptoms, and had normal serum immunoglobulin and IgE concentrations .6 B cells. A median of 3,254 total full-length, potentially functional VDJ IgE sequences were obtained per donor by ARTISAN PCR and deep PacBio sequencing, representing a median of 1.52 IgE sequences per IgE+ B cell present in the sample. In silico simulations indicated that this amount of oversampling would theoretically return a median of 78% of unique sequences  and IgA (p = 0.0017), but similar to IgM expansions (p = 0.39). Intraclonal sequence diversity was lower in IgE than in all other isotypes  in at least two B-cell aliquots , corresp applied .p < 0.0001), and less frequently IGHV4-39 than the remaining isotypes  (All isotypes had median CDR3 lengths of 15 amino acids . Unique < 0.001) .p < 0.001) and IgA . When considering all NLGM, i.e., both germline-encoded  and SHM-derived motifs, their prevalence of 27% in all IgE VDJ was significantly higher (p < 0.0001) than in IgM, IgG, and IgA  had acquired N-linked glycosylation motifs (NLGM) through SHM than IgG (16%;  and IgA . This di and IgA .In conclusion, we provide the first comprehensive inventory of near-complete peripheral blood IgE repertoires from healthy individuals through tailored methodology that lacks primer binding bias, yields full-length VDJ sequences, and detects clonal expansions by standardized parallel analysis of several aliquots . The app+ B cells have passed through germinal center reactions. The intermediate SHM load of IgE between IgM and IgG/IgA could result from direct CSR of the majority of healthy donor PBMC from IgM to IgE (+ B cells have only a limited presence in germinal centers, (The presence of SHM indicates that IgEM to IgE . Alternacenters, –27. The centers, , 16. TheThe striking lack of clonal relationships between IgE-expressing B cells and B cells expressing the other isotypes supports the important conclusion that IgE-expressing B cells emerge from qualitatively different immune responses. While the IgE repertoire has been sequenced to apparent exhaustion in our study, incomplete sampling of the other isotype compartments cannot completely exclude a low degree of clonal overlap. However, lack of clonal overlap is corroborated by a recent massive parallel sequencing study that also found virtually no clonal relationship between IgE and other isotypes in healthy donors . In contThe distinct characteristics of the normal IgE repertoire, i.e., relatively low SHM burden, lack of intraclonal sequence variation, skewed usage of IGHV genes, marked oligoclonality, retention of germline-encoded NLGM, frequent acquisition of additional such motifs, and striking absence of clonal relatedness to IgM-expressing and non-IgE-class-switched B cells cumulatively indicates its origin from qualitatively different immune responses than IgG- and IgA-expressing B cells. Like allergic individuals , 17, heaThe novel hypothesis of a specific role of N-glycosylation in expansion and maintenance of IgE-expressing B cells in non-allergic individuals warrants further studies. Ubiquitous NLGM acquisition could have a disease-specific role in selection of IgE-expressing B-cells in similarity to follicular lymphoma , primaryOur findings also support direct class switch recombination from IgM to IgE as the origin of such low-affinity IgE antibodies as demonstrated in mouse studies , 13, 19.Some, but not all , 18, 28,In comparison to allergic individuals and derivatives of high-affinity murine clones, healthy donors appear to carry IgE sequences with fewer BCR mutations , 18, 28 Overall, our results add various new aspects to the current knowledge on the IgE repertoire . Future This study was carried out in accordance with the guidelines as outlined by the LUMC Biobank Committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the LUMC Medical Ethical Testing Committee.CvB obtained samples. MK and IT obtained the data. MK analyzed the data. DA, MN, and SK provided bioinformatics support. MK and HV wrote the manuscript. All authors read and approved the manuscript.The 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.


Ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) is a synthetic bile acid-phospholipid conjugate with profound hepatoprotective and anti-fibrogenic functions in vitro and in vivo. Herein, we aimed to demonstrate the inhibitory effects of UDCA-LPE on pro-fibrogenic integrin signalling. UDCA-LPE treatment of human embryonic liver cell line CL48 and primary human hepatic stellate cells induced a non-classical internalization of integrin β1 resulting in dephosphorylation and inhibition of SRC and focal adhesion kinase (FAK). Signalling analyses suggested that UDCA-LPE may act as a heterobivalent ligand for integrins and lysophospholipid receptor1 (LPAR1) and co-immunoprecipitation demonstrated the bridging effect of UDCA-LPE on integrin β1 and LPAR1. The disruption of either the UDCA-moiety binding to integrins by RGD-containing peptide GRGDSP or the LPE-moiety binding to LPAR1 by LPAR1 antagonist Ki16425 reversed inhibitory functions of UDCA-LPE. The lack of inhibitory functions of UDCA-PE and UDCA-LPE derivatives  as well as the consistency of the translocation of UDCA-LPE and integrins, which co-fractionated with LPE but not UDCA, suggested that the observed UDCA-LPE-induced translocation of integrins was mediated by LPE endocytic transport pathway. Liver fibrosis is characterized by pathological accumulation of extracellular matrix (ECM). ECM is a collection of molecules which are secreted by cells and distributed in all organs and tissues consisting of collagens, proteoglycans, glycoproteins and glycosaminoglycans . AlthougUDCA-LPE is a synthetic bile acid-phospholipid conjugate, which has exhibited profound hepatoprotective and anti-fibrogenic functions in vitro and in vivo ,5. The cNotably, our former studies revealed that protective functions of UDCA-LPE are critically dependent on the conjugation between the bile acid and the phospholipid whereas the individual compounds UDCA or LPE showed only little efficacy in different in vitro  and in vHerein, we demonstrate the interaction of UDCA-LPE with integrins leading to integrin internalization via lipid rafts and subsequent inhibition of fibrogenic signalling. These events represent a novel mechanism of UDCA-LPE in support of its potent anti-fibrogenic effects previously observed in experimental mouse models of liver disease .The interaction with ECM leads to an autophosphorylation of FAK at Tyr397 with subsequent binding of FAK to SRC, which in turn activates SRC leading to phosphorylation of FAK at Tyr576/577 and Tyr925, which is known to be essential for its kinase activity . After pTranslocation of integrin β1 with subsequent loss of its co-localization with SRC A was assThe most prevalent integrin recognition site present in ECM contains a tripeptide motif composed of L-arginine, glycine and L-aspartic acid (RGD). RGD-containing peptides, which bind to the RGD-recognition site of integrin, inhibit their binding to the ECM. Although UDCA-LPE mediated the translocation of multiple integrins, it did not induce the translocation of integrin α1 as observed in CL48 cells . IntegriIt is known that activation of integrin signalling involves autophosphorylation of FAK at Tyr397, which leads to an interaction of FAK with SRC . With a We found that treatment of CL48 cells with UDCA-LPE or LPE was able to induce phosphorylation of b-Raf at Ser445 in the first 15 min A. It is To dissect the role of LPAR1, we further utilized an LPAR antagonist Ki16425, which was reported to disrupt the binding of LPE to LPAR1 ,20. ThusCell lysates were subjected to lipid fractionation and the levels of various integrins in 12 fractions were analysed by western blotting. A marker for lipid rafts caveolin-1 was mostly detected in fractions 2–4 of control CL48 cell lysates and in fractions 1–4 in cells treated with UDCA-LPE for 30 min A. This iThe intracellular transport of a heterobivalent ligand could be determined by one of its receptors . To inveAs effective therapeutic options against liver fibrosis are limited to date, the proposal of novel compounds which target pro-fibrogenic pathways is urgently needed. The bile acid-phospholipid conjugate UDCA-LPE has been proven to exhibit potent anti-fibrogenic functions in vitro and in vivo . In thisRecent studies have reported that TUDCA stimulates integrin-dependent phosphorylation of SRC, FAK, ERK and p38MAPK ,23. SimiThe design of heterobivalent ligands to target two different receptors has previously been used for pharmacological purposes . As a noIt has been reported that LPAR1 is localized partially in lipid rafts  and thatThe general mechanism for endocytic transport of LPE has not been well understood. LPARs are normally localized in both clathrin and caveolar endocytic microdomains and the latter is thought to respond to LPAR internalization because of LPAR co-localization with caveolin-1 in the nucleus . It has Integrins cross-talk with crucial pro-fibrogenic pathways such as TGFβ1 and PDGF signalling  and are It is well-recognized that integrin-induced signalling plays a crucial role in fibrogenesis and that the downstream proteins FAK and SRC play an essential during pro-fibrotic signalling ,41. RGD In present study, we demonstrated a possible novel pharmacological tool for integrin inhibition, where UDCA-LPE did not function as a direct inhibitor of integrins per se but as a heterobivalent ligand bridging between integrins and LPAR1. By the action of LPE/LPAR1 transporters in cells, UDCA-LPE was able to induce the translocation of integrins leading to a loss of co-localization with SRC, which resulted in dephosphorylation of FAK and SRC and inhibition of downstream fibrogenic targets. This elucidated mechanism of action renders UDCA-LPE as a drug candidate for the treatment of liver fibrosis.All reagents as well as the cultures and treatment of human embryonic liver CL48 cell line and Human Hepatic Stellate Cells (HHStec) are shown in Lysates of treated cells were subjected to western blotting analysis .Paraformaldehyde-fixed cells were subjected to immunofluorescence .2 culture flasks. After UDCA, LPE or UDCA-LPE treatment, cells were rinsed with PBS and scraped into 10 mL buffer containing 2 mM HEPES, 150 mM NaCl, 1 mM EGTA, 5 mM sodium vanadate, 10 mM sodium azide, 10 mM sodium pyrophosphate, 100 μg/mL PMSF, 1 mM sodium orthovanadate and 10 μl/mL protease inhibitor cocktail. Cells were homogenized and the lysates were centrifuged at 800× g at 4 °C for 10 min. Two mL of supernatants were incubated at 37 °C for 4 min and then incubated with 0.02 g Brij 98 at 37 °C for 5 min. The extracts were adjusted to 4 mL with 2 M Sucrose and cooled down in ice for 1 h. The extracts were gently overlaid with successive decreasing sucrose densities solutions (0.9–0.8–0.75–0.7–0.6–0.5–0.4–0.2 mol/L Sucrose) to prepare a discontinuous sucrose gradient. The gradients were centrifuged at 200,000× g in a Beckman SW 41Ti rotor for 22 h at 4 °C. Twelve fractions (1 mL for each fraction) were collected and used for western blotting and liquid-chromatography mass spectrometry (LC/MS-MS) analyses. The concentrations of total targets (proteins or lipids) in 12 fractions were normalized to 100% and the proportion or abundance of each target was reported in %. Cell lysates were subjected to sucrose density-gradient centrifugation for lipid fractionation.For each treatment group, CL48 cells were cultured in 20 × 75 cm2O with 8 mM ammonium acetate, pH 8.0 (solvent B). The flow rate was maintained at 0.2 mL/min and the gradient was started with 100% solvent A for 2.5 min, changed to 100% solvent B in 1 min, held for 16.5 min and returned to the initial condition in 3 min. Separated fractions were detected on-line by an electrospray ionization source of the tandem mass spectrometer .Following lipid fractionation, 500 μL of each fraction were extracted with 3 mL chloroform-methanol 2:1 mixture, 500 μL water and 20 μL internal standard D4-UDCA. Following centrifugation at 2500 rpm for 5 min, the lower chloroform phase was collected. Three mL of 2:1 chloroform-methanol mixture was added to the upper phase, extracted the second time and again centrifuged at 2500 rpm for 5 min. The lower phase was collected, combined with the previous chloroform phase and added to 0.4 mL 50 mM citric acid. Following mixing and centrifugation the lower phase was collected in a glass tube and the solvent was evaporated to dryness. The dried lipids were dissolved in 180 μL methanol. Concentrations of UDCA-LPE, UDCA and LPE in each lipid fraction were quantified using a liquid-chromatography mass spectrometer. The responses were calculated from the ratio of UDCA-LPE, UDCA, or LPE peak and D4-UDCA. Concentrations in nmol/mg protein were calculated from response of UDCA-LPE, UDCA and LPE used in standard curves. LC/MS-MS machine and running conditions are described in our published work . BrieflyLysates of treated cells were subjected to immunoprecipitation analysis .Statistical analysis was performed using Prism Software version 4.0 .Please see UDCA-LPE enforces internalization of integrins leading to an inhibition of downstream signalling pathways. As a possible novel mode of integrin inhibition, we described the simultaneous bivalent ligation of integrins and LPAR1 by via the LPE endocytic transport pathway. Thus, UDCA-LPE emerges as drug candidate for treatment of liver fibrosis by inhibiting integrin signalling via its internalization.


The evaporation (sublimation) of ice and snow has a major impact on global climate, since the amount of ice and snow determines Earth’s albedo. Yet, due to their complex geometry with several sharp regions which are singular for the evaporation, the precise evaporation dynamics of snow and ice crystals remains challenging to predict. Here, we study the sublimation of snowflakes and pointy ice drops. We show that the evaporation rates of water and ice drops are similar; they are both limited by the diffusive transport of the vapour. This allows us to predict ice and snowflake evaporation quantitatively by solving the diffusive free-boundary problem, which correctly predicts the rapid self-similar evolution of sharp edges and points. Beyond providing a conceptual picture to understand the sublimation of ice crystals, our results are more generally applicable to other diffusion problems such as the dissolution of salt crystals or pharmaceuticals. Ice sublimation is a common, yet little-studied, heat and mass transfer problem with climatic and industrial implications. Here, the authors show that the sublimation of ice crystals is purely diffusive and is unaffected by the underlying crystalline lattice. As such, intense ongoing research efforts are made to unravel water freezing14. Yet, the evaporation (sublimation) of ice crystals, although very common, has remained largely unexplored. Previous studies on evaporating polygonal ice crystals have shown that the crystal edges, which are sharper, were the first to recede. This has been previously attributed to defects in the crystaline structure at the crystal edge15; however no quantitative measurements or models exist of the full evaporation process.Solids generally do not evaporate in ambient air. Common sense thus suggests that ice also does not rapidly evaporate (or sublimate) on Earth. Yet, ice and snow do in fact evaporate significantly in ambient air; a thin layer of snow can be seen to disappear in typically a few days, even if the temperature stays below freezing. Understanding the formation and subsequent evolution of ice is crucial in various problems: de-icing15, the shape evolution of sublimating ice and snow crystals has nothing to do with the underlying crystal structure. We show that the rounding of sharp edges and points is the direct consequence of the singular nature of the evaporation mechanism through diffusion into the vapour phase close to regions of high curvature and describe its dynamics quantitatively for the first time. For liquid drops, the local diffusion-limited evaporation rate diverges at the contact line (a region of infinite curvature). If the drop contact line stays pinned, an outward flow in the drop then replenishes the corner region, driving the well-known “coffee-stain” effect18. As snowflakes have many sharp points, we anticipate the evaporation to be locally stronger there. However, because the mass loss due to evaporation cannot be replenished in such a solid body, evaporation at the sharp points makes them recede first, resulting in their self-similar smoothing.Here we demonstrate that contrary to crystal growth and unlike what was previously thought19. Like polygonal ice crystals15, the outward pointing protrusions of the crystal structure disappear first, suggesting that the evaporation is enhanced at sharp regions and proceeds from the outside towards the inside. To understand this dynamic evaporation pattern, we first focus on the somewhat simpler case of pointy ice droplets that exhibit an isolated singular point and subsequently consider the snowflakes , freeze them and monitor their evaporation , we deposit small water drops on a cold substrate  saturation value ρsat at the ice surface to a fixed (lower) value ρ∞ far away from the ice (see Methods). The problem then reduces to solving the coupled system of equationsρice denotes the ice density, Ω the solid boundary whose outward normal is n and vn and jn the speed and local (volume) flux of the interface, respectively, in the normal direction.The early stage of evaporation is therefore dominated by the regions of high curvature. It is important to understand this stage, since the snowflake retains regions with large curvatures almost through the whole evaporation process. We therefore model ice sublimation by solving the diffusion equation around the complex crystal geometries and because there is no replenishing flows in solids, the receding velocity at every point of the interface is completely determined by the local evaporative flux. Since the ice and water evaporation rates are similar, we assume as for liquid dropsρ(r) is mathematically equivalent to the electric potential around a charged conductor22. The strong curvature dependence we observe is thus analogous to the known electrostatic tip effect. For a charged cone of semi-angle α  < 90°, the electric field at a distance r from the tip apex scales as r = 0)26. Since the electric field is mathematically equivalent to the local evaporation rate j, we thus expect a diverging flux at the tip and thus its smoothing, as seen on our ice drops. Considering a single corner yields similar results26 and since regions of high curvature locally consist of tips and corners, the above arguments are general. However, contrary to the previous literature on electrostatic tips and pinned liquid drops, here the interface is free to move in all directions and the tip shape does not remain conical. Therefore, these arguments are only qualitative, and Eq.  parameter: the curvature κ(t). The self-similarity of the tip shape then allows us to also derive a similarity solution that gives the scaling of the curvature in time by solving Eqs. (t0 = (A/κ(0))2 denotes the distance to the singularity at the beginning of the experiment. We extract the tip curvatures at the beginning of a typical experiment and plot it in Fig. Figure ing Eqs.  and 2)  κ. Becauing Eqs. , 2 and 328Our similarity solution gives the dynamics of tip rounding and should not be confused with a possible self-similar  structure of the snowflakes, which is only a way to quantify the geometry and says nothing about the dynamics. The smoothing mechanism presented here is also original, as sharpening is usually reported for seemingly similar free-boundary problems governed by different physical mechanismstf, the ice tip has completely disappeared  yields similar results: the drop always reaches a smooth self-similar spheroidal shape whose aspect ratio depends on θ  and captures the complete shape evolution. For t  <  tf/2, the agreement is less satisfactory, since sharp regions are still present and influence the evaporation. The full evaporation dynamics can however be easily obtained with finite element simulations (see Methods). We extract half of the initial pointy ice drop shape by image processing and use it as an initial condition for the simulation. Figure We show in Fig. 29. This is not the case for the branches of the snowflakes, which are very close and interact with each other. Thus, the problem cannot be solved analytically, but we can nonetheless solve Eqs.   29. Thislve Eqs. . BecauseOur findings are relevant for studies of the ageing of snow or de-icing, but the methodology presented here is general to all free-boundary diffusive processes and can also be applied to the dissolution of solids, for instance to control the smoothness of surfaces during synthesis or to optimise crystalline shapes for drug dissolution (see Supplementary Note V ~ 5 μL) of de-ionised water on clean surfaces of various wettability (and roughness) held at temperature Ts by a water-cooled Peltier system (Anton Paar TEK 150P-C). The surfaces include the (rough) Peltier surface (contact angle θ ~ 70°), thermal grease (θ ~ 95°) and treated microscope coverslips (Menzel–Gläser #1) glued with thermal grease. Superhydrophobic surfaces (θ ~ 120°) are obtained by a soot-layer deposition, followed by a silica coating30, hydrophobic ones (θ ~ 85°) by a Parylene-C coating (SCS Labcoter PDS 2010); hydrophilic ones are glass (θ ~ 50°) or plasma-treated glass . To have a better control over our initial ice drop shape and volume, we avoid long supercooling periods where the drop evaporates by applying a cold shock. We ramp the surface temperature to its minimal value Ts = −25 °C to quickly freeze the drop, then as soon as the drop has started to freeze, the temperature is set to the desired value between −15 and −3 °C.The relative humidity (RH) is set between 1 and 7% by gently blowing dry nitrogen into an acrylic box housing the experiment (0.4 × 0.5 × 0.7 m) and monitored with a thermo-hygrometer Testo 645 (accuracy ± 2%). Pointy ice drops are produced by depositing small volumes (The time evolution of the drop profile is recorded with a Canon EOS 600D camera mounted with a high-magnification objective (Navitar). The images are then analysed using ImageJ and Matlab. The tip profile is extracted with a custom sub-pixel edge detection code and fitted with an hyperbola to measure the curvature . The full-drop profile is extracted with regular edge detection, the drop volume is measured by numerically integrating the profile (assuming axisymmetry) and the contact angle with the tangent method.Ts, RH and room temperature T0 ≈ 23–24 °C into the one used in Eqs. (ρsat(Ts) and the one far from the drop ρ∞ = RHρsat(T0) are obtained from the saturation pressure with the ideal gas law, the later being calculated using Eqs.  that we calculate with the equation in ref.31, figure 78. The ice density is taken as ρice = 918.9 kg m−3.We convert our experimental parameters  in Eqs.  and 2)  Ts, RH aing Eqs.  and (10)ρsat(Ts), we have used the usual assumptions from the liquid droplet evaporation literature, which should also be valid here as the phenomenon is very similar. We have assumed a quasi-static process, neglected convection as it only applies in the presence of wind or very large drops29, kinetic effects and the Kelvin effect, as it only applies to microscopic drops32. For ice surfaces, the presence of a microscopic amorphous layer at the surface35 could also lead to a slight change in the saturation pressure. However, as this pre-melted layer is also present in previous studies measuring ρsat, this effect should already be implicitly taken into account: if temperature induced thickness variations in the layer, change the saturation pressure here; it should do so in all previous literature, and so is automatically included in the thermodynamic variation of the saturated vapour pressure with temperature (Eq. (25). Finally, we assumed for simplicity a constant diffusion coefficient and that the ice surface was at the substrate temperature, thus neglecting thermal gradients (including the effect of evaporative cooling). This assumption being usually reasonable for liquid drops, except on heated substrates36 or for very volatile liquids37, we expect it to be even more valid for ice drops, as the ice thermal conductivity is about 4 times higher than water and 10 times higher than most organic liquids. The heat from the substrate is thus transported more efficiently to the ice surface. Moreover, the latent heat loss being proportional to the evaporation rate, since our cold ice evaporates at a much slower pace than room temperature water, we expect a much smaller evaporative cooling. At the tip though, the evaporation rate diverge for an infinitely sharp tip and thus the evaporative cooling with it. However, this occurs for a very short time only as the singularity quickly regularises itself, probably explaining why we do not need to take it into account to describe our data within our spatio-temporal resolution. We further checked the validity of the constant temperature assumption with a thermal camera (Flir C3) and found no gradients within the camera accuracy (±2 °C and ±100 μm).When deriving Eqs.  and 2)  2) with Two-dimensional 2D) axisymmetric and 3D finite element simulations are performed with the commercial software COMSOL 5.2a. We assume that evaporation is quasi-static, isothermal and purely diffusive. We thus solve the coupled Eqs.  and (2) D axisymmSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


Polyalthia longifolia, rutin, quercetin, and berberine, previously selected through molecular docking. The inhibitory potency of natural DPP-4 candidates was further determined by enzymatic, in vitro Caco-2, and ERK/PKA activation in myocyte and pancreatic cells. The hypoglycemic efficacy of the natural compounds was consecutively analyzed by single-dose and multiple-dose administration in diet-induced obese diabetic mice. All the natural-compounds could directly inhibit DPP-4 activity in enzymatic assay and Caco-2 inhibition assay, and HCD showed the highest inhibition of the compounds. HCD down-regulated LPS-induced ERK phosphorylation in myocyte but blocked GLP-1 induced PKA expression. For in vivo tests, HCD showed hypoglycemic efficacy only in single-dose administration. After 28-days administration, HCD exhibited hypolipidemic and hepatoprotective efficacy. These results revealed that HCD performed potential antidiabetic activity via inhibition of single-dose and long-term administrations, and could be a new prospective anti-diabetic drug candidate.Serine protease dipeptidyl peptidase 4 (DPP-4) is involved in self/non-self-recognition and insulin sensitivity. DPP-4 inhibitors are conventional choices for diabetic treatment; however, side effects such as headache, bronchus infection, and nasopharyngitis might affect the daily lives of diabetic patients. Notably, natural compounds are believed to have a similar efficacy with lower adverse effects. This study aimed to validate the DPP-4 inhibitory activity of clerodane diterpene 16-hydroxycleroda-3,13-dien-15,16-olide (HCD) from  AccordiDPP-4  is a serine exopeptidase that is a cleaved Xaa-Pro dipeptide from the N-terminus of oligo- and poly-peptides . sDPP-4 Polyathia longifolia, rutin and quercetin from Toona sinensis, and berberine from Coptis chinensis, whose docking scores were 79.257 (rutin), 68.290 (HCD), 65.341 (quercetin), and 65.248 (berberine), respectively, obtained the 1st, 3rd, 5th, and 6th places of 68 screened natural compounds [Generally speaking, docking is a simulation of ligand–receptor interaction using computational algorithms . Compareompounds . The datTo test which natural compounds directly inhibited the activity of the DPP-4 enzyme, 100 µM of rutin, quercetin, berberine, HCD, and two known DPP-4 inhibitors (Sitagliptin and hemifumarate) were used. When compared with original DPP-4 enzyme, all test samples showed an inhibition effect. The highest inhibition was found with HCD. Compared with hemifumarate, HCD showed higher inhibitory potency. Furthermore, compared with sitagliptin, the top three natural compounds showed lower (HCD and berberine) inhibitory potency than that of sitagliptin, which indicated natural compounds could reduce DPP-4 activity, but the inhibitory potency was not higher than clinical sitagliptin . TherefoOn the other hand, mDPP-4 was a membrane-bound protein on the enterocyte, so DPP-4 activity inhibition of the two highest-potent compounds in enzymatic tests could also be determined by using enterocytic-mimic Caco-2 cells . In 12 hCellular DPP-4 has mDPP-4 and sDPP-4 as two forms, which act as different characters within cellular response regulation . sDPP-4 Moreover, mDPP-4 could be found in the pancreatic islet with the inhibition of up-regulated insulin secretion by PKA-dependent signaling ,19. The To understand the regulating effect of selected natural compounds on blood sugar in TII DM patients, diabetic DIO mice were administered HCD, quercetin, berberine, and sitagliptin (DPP4i) combined with 4 g/kg glucose to measure blood sugar changes. After converting blood sugar levels into the area under the curve (AUC), all treated groups showed a lower AUC than the DIO mice alone, which meant lowered blood sugar levels during the same testing period . FurtherBlood sugar control effects from natural compounds could be observed with two markers: AUC of the OGTT test and HbA1c %. After 28 days of natural compound feeding, AUC of DIO mice significantly ameliorated to be lower than the controls (non-induced) A. ComparIn the pathology of diabetes, dyslipidemia has been well identified. In TII DM, dyslipidemia, especially hyperlipidemia and hypercholesterolemia, was not only induced by diabetes but also by a high fat diet . TherefoAlthough natural compounds were considered as having lower toxicity and less adverse effects than conventional agents, unsuspected toxicity in long-term administration still needs to be considered . In thisTo validate the DPP-4 inhibitory potency of HCD, berberine, rutin, and quercetin selected previously by in silico, the results of direct enzymatic alteration of selected natural compounds against DPP-4 showed inhibition effects in all compounds, with HCD being the most potent compound. In in vitro cellular assays, DPP-4-related activity and signaling change could also be observed in HCD treated in Caco-2 cells, LPS-activated ERK phosphorylation in myocytes, or PKA up-regulation in pancreatic cells. Using DIO-induced diabetic mice, HCD could decrease postprandial blood sugar regardless of single dose or long-term administration. However, HCD could not diminish HbA1c levels after long-term administration, which emphasizes HCD as not being able to decrease the highest level of postprandial blood sugar levels and eliminate blood sugar fast enough. Interestingly, HCD ameliorated DIO-induced hypertriglyceridemia and GOT activity after long-term administration. When taken together, HCD natural DPP-4 inhibitor selected from molecular docking is a compelling DPP-4 inhibitor and antidiabetic candidate.158, Arg252, and Cys257; quercetin formed four H-bonds or π–π interactions at Arg125, His126, Glu158, and Phe253; HCD formed two H-bonds at Arg125 and Arg186; and berberine only formed one H-bond at Lys55 , when a study reported that DPP-4 might regulate insulin sensitivity via degradation of GLP-1 . Up to nhibitors ,33. Fromhibitors ,35. ThesPolyathia longifolia is a febrifuge and indigestion drug in traditional Indian medication [P. longifolia has been proven to have various biological activities such as anticancer, antimicrobial, immune-modulative, anti-hypertensive, and anti-ulcer [P. longifolia bark with biological functions that pointed to two major phases: immunomodulation and anti-tumor [dication . Recentlti-ulcer . Diterpeti-tumor . Microglti-tumor . For theti-tumor , renal cti-tumor , autophati-tumor , and gliti-tumor . For breti-tumor . In renati-tumor . A previti-tumor ,46. Thisti-tumor ,48.In our previous study, rutin, quercetin, and berberine showed DPP-4 inhibitory potency while docking with DPP-4 . In the The molecular docking of natural compounds against DPP-4 was described in a previous study . Selecteg for 30 min at 4 °C, and protein concentration in suspension was quantified using a Bradford protein assay . A total of 30 μg of proteins was separated using SDS-PAGE and, subsequently, transferred to a PVDF membrane . Desired proteins were stained with appropriated 1st antibodies and HRP-conjugated 2nd antibodies. After staining, whole membranes were immersed in ECL reagent (Bio-Rad) and chemiluminescent intensity detected by a LAS-3000 imager . Chemiluminescence of each protein was normalized with chemiluminescence of GAPDH, and the protein levels were presented as the intensity ratio to the untreated controls.Cells were collected and lysed in ice-cold RIPA buffer , 50 mM Tris-HCl at pH 8.0) with protein inhibitor cocktail  at 4 °C for 60 min. Cell debris was discarded by centrifugation at 12,000 2 in water-jacket incubator (Thermo-Fisher) and detached by 0.25% trypsin/EDTA while reached 80% confluence. All experiments were carried out within 10 passages for controlling uniformity and consistency.Mouse myoblast cells C2C12, rat pancreas tumor cells AR42J, and human colorectal adenocarcinoma cells Caco-2 were obtained from the Bioresource Collection and Research Center  and American Type Culture Collection , respectively. Cells were grown in high-glucose Dulbecco’s Modified Eagle’s Medium supplied with 10% (C2C12) and 20% (AR42J and Caco-2) fetal bovine serum plus 1% penicillin/streptomycin and the medium were changed every 2 days. Cells were maintained under 37 °C, 5% CO® DPP-4/CD26 Assay Kit for Biological Samples  and following the protocol in the manual. Briefly, 50 μL of assay buffer, 20 μL of DPP-4, and 20 μL of test samples  were sequentially mixed in 96-well plate, respectively. After 30 min of incubation, 10 μL of enzyme substrate (H–Gly–Pro–pNA) was added and the optical intensity measured at 405 nm. The DPP-4 activity treated with hemifumarate was set as 100%, the tested samples were measured with hemifumarate and the data presented as the ratio to hemifumarate.In vitro enzymatic assay of DPP-4 was carried out by Enzo5 cells/wells of AR42J cells was seeded into a 12-well plate and incubated overnight for confluence. Then, cells were incubated with 1 nM of GLP-1, exendin-4 (Ex-4), natural compounds with GLP-1, or natural compounds with Ex-4 for 48 h, respectively. Finally, PKA levels within AR42J were determined by Western blotting. LPS-induced ERK-phosphorylation followed the above protocol with slight modification. A number of 5 × 104 cells/wells of C2C12 was used in this study. After incubating overnight, 10 ng/mL of LPS was added for 10 and 30 min, respectively, followed by culturing with natural compounds for 12 h. Phosphorylated-ERK was assayed by Western blotting.In vitro cellular DPP-4 inhibition assay could be divided into 3 assays: ERK phosphorylation inhibition in LPS-stimulated muscle cells, PKA activation in incretin-induced pancreatic cells, and expression inhibition in Caco-2 cells. The inhibition of DPP-4 expression in Caco-2 cells was performed by Western blotting. Caco-2 cells were inoculated into a 12-well plate followed by natural compounds’ treatment for 12, 24, and 36 h, respectively. After treatments, DPP-4 levels and PKA activating inhibition were determined by Western blotting. A number of 1 × 10w/v) diet for 14 weeks; control group was fed with a normal diet. At week 10, mice were treated with an oral glucose tolerance test (OGTT) and an insulin intolerance test for the determination of DM type. If the blood sugar level at 120 min after gavaging was higher than 200 mg/dL, mice were categorized as insulin intolerant. When the insulin-intolerant mice failed the insulin intolerance test, the mice could be diagnosed as TII DM mice.Six-week-old male ICR mice were obtained from the National Laboratory Animal Center  and kept in controlled environmental conditions at room temperature (22 ± 2 °C) and with humidity (50 ± 10%). The 12 h light/dark cycle were maintained throughout the study. Mice had free access to food and water and were maintained on a standard laboratory diet. Animal experiments were approved by the National Dong-Hwa University Animal Ethics Committee  and were used according to the “Guide for the Care and Use of Laboratory Animals” of National Dong-Hwa University. Diet-induced obese (DIO) mice were treated with a high-fat and high-fructose diet. ICR mice were divided into 2 groups: DIO mice were fed with high-fat  and high fructose  prior to oral gavage (p.o.) with 4 g/kg glucose solution. After p.o., blood sugar was continuously measured at 30, 60, 90, and 120 min after gavaging. All data in each group were collected and plotted with gavaging time to calculate the area under curve (AUC). Insulin intolerance testing used the same protocol with OGTT. After p.o. glucose, mice were immediately intraperitoneal (i.p.) injected 0.8 IU/kg Bwt insulin solution .To determine hypoglycemic efficacy in a single dose, OGTT in DIO mice was employed. OGTT was tested on DIO mice with the same protocol as in the previous section. After p.o. glucose, mice were immediately treated p.o. with natural compounds and blood sugar levels tested.Long term administration was carried out in DIO mice. Diabetic DIO mice were allotted to treated groups for 6 mice and treated p.o. with natural compounds once per 7 days for 28 days. Body weight was measured every 7 days. At the end of treatment, OGTT was carried out to check insulin tolerance.At the beginning and end of treatment, 500 µL of whole blood was collected by piercing the cheek and separating into 2 parts: 100 µL mixed with 3.2% sodium citrate to measure glycated hemoglobulin (HbA1c) immediately; 400 µL incubated at room temperature for coagulation and then centrifuging at 4000 rpm for 10 min to collect serum using total triglyceride (TG), total cholesterol (CHO), glutamate oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT) tested by an automatic analyzer .p < 0.05. All statistical procedures were performed with GraphPad Prism version 7.0 .All data were expressed as means with standard deviations (mean ± SD) and the data were analyzed using one-way ANOVA with a Dunnett’s test. The level of statistical significance was set at When taken together, HCD is first evident in showing DPP-4 inhibitory activity and blood sugar lowering efficacy in short-term and long-term administration. Combining with α-amylase/α-glucosidase, inhibition potency having been previously demonstrated, HCD poses a high potential for becoming an antidiabetic drug candidate. The antidiabetic potency of HCD has been under-estimated in research and it worthwhile seeking the impact of a clinical trial.


Under the condition of this study, heat-treated nickel–titanium (NiTi) files with smaller cross-sectional area, fewer contact points, and reciprocating movements resulted in a lower screw-in effect. This study compared the maximum screw-in forces of various instruments during their movements. Forty simulated canals in resin blocks were randomly divided into four groups (n = 10): ProTaper Universal F2, ProTaper Gold F2, WaveOne Primary, and WaveOne Gold Primary. To standardize a lumen size, all artificial canals were prepared with ProTaper Universal F1. The rotation speed was set at 350 rpm with an automated 4 mm pecking motion at a speed of 1 mm/s. The pecking depth was increased by 1 mm for each pecking motion until the file reached the working length. During instrumentation, screw-in forces were automatically recorded by customized software. Maximum screw-in forces were analyzed by one-way ANOVA and Tukey’s post hoc comparison with the significance level at 0.05. WaveOne Gold files generated the lowest maximum screw-in forces, followed by ProTaper Gold, WaveOne, and ProTaper Universal ( Because of superior flexibility and higher torsional fracture resistance of nickel–titanium (NiTi) instruments compared to those of stainless-steel (SS) instruments, NiTi instruments are widely chosen for the root canal treatment . ShapingGeometric features and heat treatment could influence the mechanical properties ,5,6 and This screw-in phenomenon has been reported to occur frequently when the cutting tip of the rotary instruments are active and have sharp fluted edges ,14. OverVarious kinds of engine-driven NiTi instrument systems made from different alloys and geometric designs are marketed in contemporary endodontics. Few studies have examined the relationship between the features of NiTi files and the screw-in tendency. Previous studies have demonstrated that the screw-in tendency is affected by cross-sectional design and pitch length rather than shaping motion ,20. The Therefore, the aim of this study was to compare the screw-in forces of various instruments with different heat treatments and kinetics during their movements. The null hypothesis was that the heat treatment, geometric properties, and kinetics of endodontic files will not influence the screw-in force during simulated root canal preparation.Four file systems made of different alloys and with different kinetic movements were tested: Two continuous rotation systems  and ProTaper Gold F2  and two reciprocating systems  and WaveOne Gold Primary ). PTU is made of conventional NiTi alloy; WOP is made by using M-wire; PTG and WOG are made with “Gold-Wire” . All instruments have a tip size of ISO #25 with a different apical taper: PTU, PTG, and WOP have 8% apical taper; WOG has 7% apical taper. Their cross-sectional area at D4, helical angle, and pitch length were measured using 30× magnification under a stereomicroscope  a–d.Forty simulated J-shaped root canals in resin blocks (Dentsply Sirona) were used in this study (n = 10). The curvature of root canal was determined to 35° according to the Schneider method , and theA custom-made test device   was fabrp < 0.05. All statistical analyses were performed using SPSS version 22 for Windows .The maximum screw-in forces during the instrumentation procedures were checked for the normality of distribution using the Kolmogorov–Smirnov test. Then, the data were analyzed using the one-way analysis of variance and Tukey’s post hoc comparison test. The level of significance was set at p < 0.05).The mean and standard deviation values of screw-in forces for different tested endodontic files are shown in Previous studies have reported that the geometric features and metallurgical properties of the NiTi file systems have an effect on their mechanical performances ,25,26,27The resin blocks with simulated root canals were used for the standardization of experimental condition and might have a methodological limitation. Even though experimental conditions were standardized by the use of simulated resin canals, the properties of resin material are different from those of natural dentin. Although extracted natural teeth could be used to simulate a clinical condition, the screw-in forces generated during root canal shaping may be highly affected by the anatomic deviations such as canal curvature and lumen size shown in natural teeth . A previ2 at a 4 mm level) and a unique cross-sectional geometry  similar to a slender rectangular and one- or two-point contacts with the dentinal wall during root canal preparation. These geometric features of WOG might have contributed to generating the lowest screw-in forces during instrumentation.Both the geometric design and manufacturing process (heat treatment) of instruments may affect the flexibility and the reaction forces as well as the screw-in effect . In the In contrast, PTU showed the highest screw-in forces. PTU is made of conventional NiTi alloy and has a triangular cross-sectional area designed to cut root dentin with three-point symmetrical contacts. PTU also has a larger taper (0.08 taper for F2) compared with WOG (0.07 taper for Primary). These characteristics increase its rigidity and may produce a higher screw-in effect.In the present study, PTG showed the lower maximum screw-in force compared with PTU, despite these two file systems having similar geometric features. This might be explained by the characteristics of Gold-Wire, which might share thermomechanical properties with CM-wire, and cause the least amount of canal transportation due to its higher flexibility and minimized reaction force ,31. The Manufacturers have claimed that reciprocating movements could reduce the screw-in effect . A transAlthough the screw-in tendency is harmful to root canal preparation, to date few studies have reported the screw-in effect related to NiTi instruments ,19,20. CUnder the limitations of this study, heat-treated NiTi files (WOG and PTG) generated lower screw-in forces compared to M-wire (WOP) and conventional NiTi files (PTU). WOG, which has a smaller cross-sectional area with fewer contact points, generated lower screw-in forces. Reciprocating movement adopted by WOP and WOG may also have effect on the decreased screw-in effect.


The abnormal accumulation of amyloid-β (Aβ) in the central nervous system is a hallmark of Alzheimer’s disease (AD). The regulation of the processing of the single- transmembrane amyloid precursor protein (APP) plays an important role in the generation of Aβ in the brain. The phosphorylation of APP and key enzymes involved in the proteolytic processing of APP has been demonstrated to be critical for modulating the generation of Aβ by either altering the subcellular localization of APP or changing the enzymatic activities of the secretases responsible for APP processing. In addition, the phosphorylation may also have an impact on the physiological function of these proteins. In this review, we summarize the kinases and signaling pathways that may participate in regulating the phosphorylation of APP and secretases and how this further affects the function and processing of APP and Aβ pathology. We also discuss the potential of approaches that modulate these phosphorylation-signaling pathways or kinases as interventions for AD pathology. ApoE ε4 has been suggested to be a major genetic risk factor for sporadic AD [ApoE ε4 may have a 5–8-fold increase in the risk of developing AD [APP, PS1 and PS2 could cause autosomal dominant familial AD, which usually causes disease symptoms before the age of 65 [phosphatidylinositol binding clathrin assembly protein (PICALM) for lipid metabolism and complement receptor type 1 (CR1) and triggering receptor expressed on myeloid cells 2 (TREM2) for immune regulation) have also been associated with the risk of developing AD [Current epidemiological studies estimate that there are approximately 45 million people suffering from dementia . With thradic AD . Individoping AD . Mutatioge of 65 . Genome-oping AD . To dateoping AD . The fouoping AD .The phosphorylation of proteins is a fundamental posttranslational modification whereby target molecules are covalently modified at serine, threonine or tyrosine residues by the addition of phosphate groups . The regAPP gene is located on chromosome 21. The alternative splicing of the APP gene produces three isoforms: APP695, APP751, and APP770 (the numbers indicate the number of amino acid residues). The extracellular domain of APP contains two independently folded E1 and E2 subdomains and a juxtamembrane domain [APP is a type-I single transmembrane protein with a long extracellular domain and a short cytoplasmic domain . The APPe domain . The trae domain . The phye domain . Upon gee domain . When APe domain . It has e domain . APP maye domain .The cleavage of APP by α-secretase does not produce Aβ proteins in neurons because the cleavage site of α-secretase is located within the Aβ domain (the peptide bond formed by Lys16 and Leu17 in the numbering of Aβ40) . Chen and co-workers proved that leucine-rich repeat kinase 2 (LRRK2) is capable of interacting with and phosphorylating APP at T668 residues. Following cleavage, the phosphorylated AICD translocates to the cell nucleus with elevated transcriptional activity and exacerbates LRRK2-mediated neurotoxicity and dopaminergic neuronal loss . Most of2+/calmodulin-dependent protein kinase II (CaMKII) as the catalytic enzymes of the phosphorylation process [653YTSI656 motif, which is involved in the trafficking and sorting of APP [Although the T668 residue appears to be the most extensively studied phosphorylation site on the APP, the first reported phosphorylation sites were T654 and S655, which were identified by Gandy et al. using synthetic peptides corresponding to the sequence of APP645–661 [ process . Isohara process ,58,90,91g of APP . Using fg of APP . A receng of APP . The outg of APP . ROCK1 ig of APP . Hendersg of APP . Aβ oligg of APP . ROCK2 ig of APP . ROCK2 cg of APP .S675 is another serine residue that has been detected to be phosphorylated in the brains of AD patients, but its function was only recently clarified. Lee et al. reported that Polo-like kinase 2 (Plk2) is able to phosphorylate both T668 and S675 residues . Plk2 is682YENPTY687 motif has important effects on the trafficking and metabolism of APP [There are three tyrosine residues  within the cytoplasmic domain of APP. The Y653 residue has been observed to be critical for the polarized sorting of APP to the cell surface . The mosm of APP ,100,101.m of APP ,103. Thim of APP ,68. Nervm of APP ,68. Aftem of APP . Growth m of APP ,105. Zhom of APP . A recenm of APP . The incm of APP . In addim of APP . Therefom of APP . The Y68m of APP . Rebelo m of APP ,71. In cm of APP ,71. In am of APP .It is evident from these findings that the phosphorylation of APP at its cytoplasmic domain has a significant effect on its proteolytic cleavage and functions. These effects can be attributed directly to the modulation of trafficking and subcellular localization of APP after phosphorylation, or indirectly to the influence on the interaction between APP and its binding partners . It is r32P isotope labeling experiments [The phosphorylation of the extracellular domain of APP is rare, while the serine residues in the extracellular domain of APP can also be phosphorylated, as demonstrated by eriments . Two sereriments . Walter eriments , and thaeriments .The α-secretase mediated cleavage of APP is mainly performed by membrane bound proteases of the ADAM family. In particular, ADAM10 and ADAM17 are the major catalytic enzymes of this process ,111. ADAThe aberrant activation of BACE1 has been documented both in normal aging and in AD ,117. In There are four subunits in the γ-secretase complex, each with distinct functions and regulatory pathways. PS is the catalytic subunit of γ-secretase and is rich of serine, threonine, and tyrosine residues, suggesting that it can be phosphorylated by kinases . The phoAs has been summarized before, the phosphorylation signaling in APP processing involves a variety of kinases, phosphorylation sites, and substrate proteins and has divergent effects on the function and proteolytic processing of APP. Since kinases are broadly involved in regulating different biological events, it might be challenging to specifically address the phosphorylation of proteins involved in APP metabolism in the brain without influencing other physiological functions of the kinase. The BBB poses additional challenges for drug candidates to enter the target brain regions . Many kiThe phosphorylation of APP at T668 is believed to be an important clinical feature in brains of AD patients and has been recognized to accelerate Aβ generation and plaque formation. It might be beneficial to block this phosphorylation event in the brain in order to ameliorate AD pathology. The mutation of T668A in APP could prevent the phosphorylation of APP in vivo and rescue memory and synaptic plasticity deficits in a familial AD mouse model . InsulinAD is a complex neurodegenerative disease with a multifaceted pathogenesis. APP processing is a fundamental aspect both under physiological and pathological conditions. Although the enzymatic cleavage pathways of APP are relatively well-defined, the regulation of APP processing by different signaling pathways is not completely understood. Phosphorylation/dephosphorylation represent a key mechanism of the modulation of protein functions and structures. In AD, the phosphorylation of APP has been established at multiple levels, including both human and animal studies. The close link between the abnormal phosphorylation of proteins and AD pathology emphasizes that phosphorylation signaling pathways might be dysregulated during disease progression. Some of the phosphorylation sites, such as the T668 residue, can be phosphorylated by several kinases. Phosphorylation may directly influence the subcellular distribution of APP and therefore the proteolysis, or indirectly affect the binding between APP and other proteins and then induce further changes . Underst


Histamine-releasing activities on human basophils have been studied as potential allergy-causing agents for four decades. An IgE-dependent histamine-releasing factor (HRF) was recently shown to interact with a subset of immunoglobulins. Peptides or recombinant proteins that block the interactions between HRF and IgE have emerged as promising anti-allergic therapeutics, as administration of them prevented or ameliorated type 2 inflammation in animal models of allergic diseases such as asthma and food allergy. Basic and clinical studies support the notion that HRF amplifies IgE-mediated activation of mast cells and basophils. We discuss how secreted HRF promotes allergic inflammation in vitro and in vivo complex disease settings. Activation of mast cells and basophils via high-affinity IgE receptors (FcεRI) on the cell surface plays an essential role in allergic reactions. Multivalent allergens induce the aggregation or cross-linking of IgE-bound FcεRI to trigger their activation . ActivatTPT1 gene on chromosome 13. Although numerous single nucleotide polymorphisms (SNPs) are associated with allergic diseases, no genetic associations with gene expression (eQTLs) are found in the TPT1 locus (http://dicew-database.org). Similar to antigen/IgE-mediated activation, HRF induces not only histamine release, but also IL-4 and IL-13 secretion from human basophils and IL-13 and TNF secretion from murine mast cells [+, can prime basophils in response to HRF, but other IgE molecules, termed IgE−, are unable to do so [+ vs. IgE− was discovered long before the molecular cloning of HRF, and several possibilities exist to explain the heterogeneity of IgE molecules: 1) structural differences in the constant regions of IgE, for example, by differences in glycosylation or alternative mRNA splicing at the ε chain 3′ terminal region [+ being an HRF-specific IgE antibody, that is, HRF acting as an IgE autoantigen; 3) IgE+ reactivity due to the presence of anti-IgE antibodies in the serum.Cytokine-like factors able to activate basophils in body fluids of allergic patients have been studied for many years . Severalst cells ,16. Despst cells ,18,19,20to do so . The dicl region ; 2) IgE++ vs. IgE− may be caused by differences in IgE variable region subgroups [+ reactivity is related to glycosylation of IgE [+ or with IgE− [H and VL regions might contribute to the IgE+ reactivity. In light of recent revelations regarding IgE glycosylation [In contrast to an earlier report suggesting that HRF does not bind to IgE , Kashiwaubgroups . Howevern of IgE  was not ith IgE− . Despitesylation , the potE. coli can activate murine mast cells [HRF is constitutively secreted as a monomer, a disulfide-linked dimer, and higher molecular weight oligomers. Crystal structures of HRF monomers from various species and a homodimer of human HRF have been solved. The homodimer is made by a disulfide bond through a Cys172-Cys172 linkage between two monomers ,29. Kim st cells . GST-HRFst cells  but alsost cells ,16. It ist cells . The widAllergic diseases such as atopic dermatitis, food allergy, asthma, and allergic rhinitis are type 2 inflammatory diseases in allergen-sensitized individuals with organ-specific or systemic disease susceptibility ,32,33. TSites within the HRF molecule led to the discovery of HRF sequence-based competitive inhibitors, N19 and H3 peptides, as well as a monomeric mutant HRF-2CA, all of which blocked HRF–Ig interactions without affecting intracellular functions of TCTP. Administration of these inhibitors drastically reduced type 2 inflammation in mast cell-dependent murine models of atopic asthma and immediate hypersensitivity of the skin. Intranasal administration of recombinant HRF into naïve mice caused lung inflammation in an FcεRI and mast cell-dependent manner . Thus, tSaussurea lappa Clarke, which is able to bind to HRF dimers, was reported to suppress ovalbumin-induced airway inflammation [Another interesting drug candidate is a 7-mer peptide, called dTBP2. It was identified by phage display as a peptide more strongly bound to HRF dimer than to monomeric HRF . dTBP2 aammation . HoweverAtopic dermatitis is a heterogeneous disease in terms of the pathogenic role of the IgE–FcεRI axis ,56. InteChronic idiopathic urticaria (CIU) or chronic spontaneous urticaria is a disease of itchy red skin or skin colored hives with no known cause lasting for six weeks or more. IgG autoantibodies against IgE or FcεRI may contribute to CIU pathogenesis in 30%–40% of the patients . ActivatBMPR2). Excessive proliferation of pulmonary vascular endothelial cells is seen in this disease caused by an imbalance between cell proliferation and apoptosis. Increased plasma and lung levels of HRF associated with exosomes derived from endothelial cells were found in PAH patients compared to normal subjects [Pulmonary arterial hypertension (PAH) is a rare, but often lethal disease characterized by a sustained increase in pulmonary arterial pressure and severe vascular remodeling. Heritable PAH commonly involves mutations in bone morphogenetic protein receptor type II (subjects ,64. The subjects ,63. Thessubjects . No defi+/−Ldlr−/−Apobec1−/− mice contain fewer macrophages and more apoptotic cells compared to TCTP+/+Ldlr−/−Apobec1−/− mice [It is not easy to assign a particular pathogenic role to the secreted HRF molecules separate from the intracellular TCTP molecules. Targeting HRF is a promising approach toward prevention of allergic diseases such as food allergy and asthma ,30,65. H−/− mice . Transge−/− mice . They sh


We find strengthening of westerly wind during the austral summer (between 50°S to 62°S) facilitated northward advection of a cool and fresh layer. Also, the strong westerly wind cools the upper ocean due to net heat loss from the ocean surface. The combined effect of northward advection of cold fresh layer and net heat loss from the surface, favours sea-ice expansion in the subsequent seasons, in the IOA region, north of 62°S. However, sea-ice retreat was observed near the Kerguelen Plateau, due to upper ocean warming, and hence a non-annular pattern of sea-ice extent in the IOA was observed.In the backdrop of global warming, Antarctic sea-ice variability showed an overall expansion with the regional heterogeneity of increasing and decreasing patterns. Analysis of satellite derived sea-ice extent, during 1979 to 2015, in the Indian Ocean sector of Antarctica (IOA) revealed expansion of 2.4±1.2% decade The Southern Ocean plays a crucial role in the Earth’s climate system and its sea-ice coverage is a key indicator of climate change that modulates the albedo, air-sea exchange of heat, fresh water, carbon, ocean-atmospheric circulation, Antarctic ecosystems, and biogeochemical cycle ,2. The iin-situ observations [The data sets used in this study are: (i) Monthly sea-ice extent and concentration (1979–2015) from the Scanning Multichannel Microwave Radiometer (SMMR), the Special Sensor Microwave Imager (SSMI), and the Special Sensor Microwave Imager Sounder (SSMIS) in polar stereographic projection at 25×25 km spatial resolution acquired from the National Snow and Ice Data Center (NSIDC), Colorado; (ii) National Oceanic and Atmospheric Administration (NOAA) monthly optimum interpolated sea surface temperature (OI SST) v2 (1982–2015) at a spatial resolution of 1°×1° constructed after blending of Advanced Very High Resolution Radiometer (AVHRR) and rvations ; (iii) Qrvations ; (vi) Grrvations ; and  according to a two-tailed t-test. The water masses such as Antarctic surface water (AASW), Antarctic intermediate water (AAIW), and circumpolar deep water (CDW) are characterised after following the standard methodology [The sea-ice extent and concentration are derived from passive microwave brightness temperature using the National Aeronautics and Space Administration (NASA) algorithm for the period of 1979–2015. The sea-ice extent is computed by summing-up the areas of all pixels having at least 15% of sea-ice concentration in the IOA between 20°E to 90°E . The timor ORAS4 ,36 and Eor ORAS4 . In ordeor ORAS4 . Ocean chodology ,40.http://esa-cci.nersc.no) is available, e.g. from the Copernicus data portal for Envisat: http://catalogue.ceda.ac.uk/uuid/b1f1ac03077b4aa784c5a413a2210bf5 and CS2: http://catalogue.ceda.ac.uk/uuid/48fc3d1e8ada405c8486ada522dae9e8. The sea-ice area ‘A’ derived from the combined Eumetsat OSI-SAF / ESA-SICCI-2 AMSR-E / AMSR2 sea-ice concentration (SIC) data set  [http://nsidc.org/forms/nsidc-0116_or.html) with monthly temporal resolution. For meridional fluxes at gates 1 and 2 we only use the v-component of the ice motion; for zonal fluxes at gates 3 to 6 we only use the u-component of the ice motion. We co-locate the sea-ice motion vectors, which are on the NSIDC polar-stereographic grid with tangential plane at 70°S, onto the EASE2.0 grid used for the SIC and sea-ice thickness data. We only use cases with SIC > 70% in accordance with the settings of the sea-ice freeboard retrieval [In order to verify whether the observed increase in sea-ice extent and concentration are compensated by the loss of sea-ice volume, we compute the sea-ice freeboard flux, i.e., sea-ice freeboard ‘F’ times sea-ice area ‘A’ times the relevant component of the sea-ice motion vector ‘v’ for the period 2002 through 2017 for the gates denoted in 7bc041e) . SICCI-57bc041e)   with regional heterogeneity that comprises of increasing and decreasing pattern in different sectors (-1), followed by autumn (4.0%±1.6 decade-1), winter (1.9%±0.9 decade-1) and spring (1.7%±1.2 decade-1). We note that the absolute trend values for IOA are relatively small in relation to the size of one grid-cell: ~25 km x ~25 km = 625 km2; hence, the annual trend of 3.0x103 km2 corresponds to 4–5 grid cells. Overall, we find an increasing trend of sea-ice concentration and extent in the IOA, which is associated with sea surface cooling  and thereby increasing the surface current (up to 0.35±0.08 cm s-1 decade-1) in the austral summer (between 50°S to 62°S) suggesting northward transport of these comparably fresh, cold water from the southern boundary of the Antarctic circumpolar current  cooled significantly, except the warming and corresponding sea-ice reduction near the south Kerguelen Plateau, and coastal regions of the Prydz Bay, Mawson, Prince Harald. The remainder water column below ~100 m warmed significantly, which has been detected earlier and linked to the advection of CDW that delivers warm water onto the ice shelves ,57,66. IS1 Table(DOCX)Click here for additional data file.S1 FigSea-ice freeboard fluxes are computed across 64°S latitude (gates 1 and 2), at the western (gates 3 and 4 at 20°E) and eastern (gates 5 and 6 at 100°E) entry to the Indian Ocean sector. The red line at ~53°E separates between western IO = Weddell East and eastern IO = East Antarctic West.(TIF)Click here for additional data file.S2 FigYears and vertical dashed lines denote the beginning of the respective calendar year.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigThe dashed lines are the running mean of 10-year data for different seasons; indicates significant trend towards high-index polarity during the austral summer (dashed red line) and autumn (dashed black line).(TIF)Click here for additional data file.S6 FigThe data are obtained from the Technische Universität Dresden, Germany, derived from the Gravity Recovery and Climate Experiment (GRACE) satellite.(TIF)Click here for additional data file.S7 Figa) summer, (b) autumn, (c) winter, and (d) spring, over the period of 1979–2015. Right panel figures indicates the trends of salinity for (e) summer, (f) autumn, (g) winter, and (h) spring. Only the significant trend values (p < 0.05) are considered to plot the figures according to a two tailed t-test and the dots are marked to show the observations with the significant trends. The contours indicate the climatology of temperature and salinity overlaid on the color shaded trend maps.The European Centre for Medium range Weather Forecast (ECMWF)’s EN4 data shows the cross section of the zonally averaged (20°E to 90°E) trends of potential temperature for ((TIF)Click here for additional data file.S8 Figa) summer, (b) autumn, (c) winter, and (d) spring. The optimum interpolated SST (OI SST), Interim SST, Ocean Reanalysis System 4 (ORAS4) SST and EN4 SST data are used for the analysis. EN4 SST (pink line) shows large deviations (probably uncertainties) compared to other datasets, particularly during austral autumn and winter.Sea surface temperature (SST) anomaly computed from multiple data showed robust feature of sea surface cooling over the Indian Ocean sector of Antarctica (averaged from 20°E to 90°E and 73°S to 55°S), for ((TIF)Click here for additional data file.S9 Fig-1) overlaid on the sea-ice concentration climatology (1979–2015), for (a) summer, (b) autumn, (c) winter, and (d) spring, generated using ERA-Interim reanalysis data. Only the significant trend values (p < 0.05) are contoured according to a two tailed t-test. Overall, the increasing pattern of sea-ice concentration is evident in the Indian Ocean sector of the Southern Ocean except the reduction in sea-ice near the south Kerguelen Plateau (SKP), and coastal regions of the Prydz Bay (PB), Mawson (MS), Prince Harald (PH).Sea-ice concentration trend contours (% decade(TIF)Click here for additional data file.S10 Figa) summer, (b) autumn, (c) winter, and (d) spring, over the period of 1979–2015, computed from the European Centre for Medium range Weather Forecast (ECMWF)’s Ocean Reanalysis System 4 (ORAS4) data. Enhanced warming is observed throughout the water column both at the surface and subsurface with a concurrent reduction in sea-ice as shown in Figs The cross section of the zonal temperature trend at 85°E (south Kerguelen Plateau) for ((TIF)Click here for additional data file.