Cryopreservation technology has developed into a fundamental and important supporting method for biomedical applications such as cell‐based therapeutics, tissue engineering, assisted reproduction, and vaccine storage. The formation, growth, and recrystallization of ice crystals are the major limitations in cell/tissue/organ cryopreservation, and cause fatal cryoinjury to cryopreserved biological samples. Flourishing anti‐icing materials and strategies can effectively regulate and suppress ice crystals, thus reducing ice damage and promoting cryopreservation efficiency. This review first describes the basic ice cryodamage mechanisms in the cryopreservation process. The recent development of chemical ice‐inhibition molecules, including cryoprotectant, antifreeze protein, synthetic polymer, nanomaterial, and hydrogel, and their applications in cryopreservation are summarized. The advanced engineering strategies, including trehalose delivery, cell encapsulation, and bioinspired structure design for ice inhibition, are further discussed. Furthermore, external physical field technologies used for inhibiting ice crystals in both the cooling and thawing processes are systematically reviewed. Finally, the current challenges and future perspectives in the field of ice inhibition for high‐efficiency cryopreservation are proposed. This work describes the fundamental mechanisms of ice injury during cryopreservation, and introduces the state‐of‐the‐art ice‐inhibition materials and strategies, both in the cooling and thawing processes, for high‐efficiency cryopreservation. Future perspectives and challenges are also proposed to motivate the development of cell, tissue, and organ cryopreservation and offer bright new inspiration for cryobiology. Cryopreserved cells or tissues not only maintain normal structure and function integrity after thawing from cryogenic temperatures, but also allow for further clinical application and fundamental research. Until now, the basic theories, cryogenic devices, cryopreservation strategies, and novel cryoprotectants (CPAs) for cryopreservation have made a breakthrough in the field of cryobiology, thus promoting considerable development of numerous biomedical applications for cryopreservation, including assisted reproduction, stem cell therapies, regenerative medicine, tissue engineering, biological sample banking, and the development and research of drugs. For example, with recent advances in flourishing stem cell‐based medicine, the demand for stem cells is sharply increasing. Hence, achieving high‐quality and high‐efficiency storage of stem cells is vital to overcome the current supply–demand imbalance. Additionally, another typical application area is human fertility. Recently, the risk of infertility in young women has been increasing due to occasional disease, aggressive medicine use, and external pressures. The reproductive diseases occurring in young men worldwide seriously affect their fertility continuity. Therefore, the cryopreservation of egg cells, sperm, ova, and embryos are crucial for human reproduction. In summary, the primary and indispensable cryopreservation science for various biological samples  is vital for clinical applications and scientific research in the field of biomedical engineering.Cryopreservation is a basic and important technique to achieve long‐term storage of organs, tissues, cells, and other biological materials by using a very low temperature (at −80 or −196 °C).1, 2 Among these injuries, the formation, growth, and recrystallization of ice crystals are fatal for cryopreserved samples, causing primary problems and limitations for realizing efficient cryopreservation. It is well known that liquid water is essential for maintaining the structure and function integrity of living cells. However, the inevitable water‐to‐ice phase transition (intracellular and extracellular ice) at the freezing temperature usually leads to cells sustaining mechanical ice damage. The elevated solute concentration due to ice formation also results in irreversible injury to cells after undergoing freeze–thaw cycles. To date, cryopreservation strategies are mainly divided into the slow freezing and vitrification methods. For the most common and traditional slow‐freezing techniques, despite allowing low‐CPA solution, ice injury both in intracellular and extracellular solutions is a crucial factor for cell survival. To minimize ice damage, many cryogenic researchers have put great effort into optimizing the parameters of cryopreserved conditions, including the concentrations of CPA, cooling rates, cooling temperature, and warming rates. Owing to the advantage of ice‐free solution in the process of freezing, the vitrification method has attracted considerable attention, and is regarded as the most promising way to achieve organ cryopreservation in the future. However, the main limitation of vitrification is the formation of ice nucleation and devitrification in the warming process, which causes fatal injury to cryopreserved samples. It is self‐evident that the formation and growth of ice crystals, as well as the accompanying mechanical damage, are the dominant reasons for the destruction of the function and structure of cryopreserved biological samples. Hence, the control, restriction, and elimination of ice crystals are fundamental and crucial scientific problems in the field of cryopreservation.In the process of cryopreservation, a variety of chemical and physical damage occurring in the procedures of freeze–thawing are the main destruction mechanisms of cryopreserved biological samples.26 Amo Nevertheless, to the best of our knowledge, a systematic and comprehensive review of ice control for cryopreservation has not been found. To this end, as depicted in Scheme With the rapid development of biology, chemistry, and material research, innovative materials and biotechnology tools are utilized to suppress the formation and growth of ice crystals; this offers a revolutionary improvement to enhance cryopreservation science.45 Nev2Figure As a fundamental and important approach to storing biological specimens, cryopreservation can effectively reduce metabolism and offer vital support for various biological applications. The basic cryopreservation procedures and cryoinjuries that occur during the cooling and warming processes are summarized in  which can be defined as slow freezing and vitrification  in the thawing process promotes the transformation of some small ice crystals and liquid water into larger ice crystals. Currently, inhibition of the formation and growth of ice crystals during the warming process is of great significance for improving the efficiency of cryopreservation, and remains a research hotspot in the field of cryogenic storage. Overall, ice recrystallization is a complex process, and is closely correlated with different warming parameters. Warming rates and temperatures are vital influencing factors for the nucleation and growth of extracellular ice.Extracellular ice not only leads to mechanical damage to the cell, but also causes an accompanying increase in solute concentration, resulting in osmotic injury.49 As  With respect to intracellular ice formation, the main theories that elucidate the intracellular formation mechanisms include pore theory, surface‐catalyzed nucleation (SCN) theory, volume‐catalyzed nucleation (VCN) theory, and cell membrane damage theory. In addition to these theories, numerous experimental data have demonstrated that intracellular ice is affected by many factors, such as cooling rates, permeability to water, intracellular supercooling, the integrity of cell membranes, and extracellular ice. Typically, the establishment of a prediction model and equations for intracellular ice, developed by Mazur, demonstrated that the formation of intracellular ice mainly depended on the cooling rates and the permeability of water to cells, which provides insight into the determining factors for cell survival. Likewise, in the process of warming, variable parameters influence intracellular ice nucleation and growth. As shown in Figure  Furthermore, it has been proved that intracellular ice produced more serious recrystallization under slower warming rates at the same temperature condition, implying that the faster warming rate is helpful in inhibiting recrystallization and weakening intracellular ice injury. Indeed, intracellular ice can result in a fatal injury to cryogenic cells in freeze–thaw cycles. Thus, exploring the formation mechanism of intracellular ice and eliminating accompanying injury to organelles remains a research priority in the field of cryobiology.Compared with extracellular ice, intracellular ice occurs in the condition of rapid cooling, as a result of the frozen intracellular water. In order to overcome major intracellular‐injury obstacles for cryopreserved cells and tissues, understanding the underlying intracellular ice‐formation mechanism and controlling intracellular ice via experiment and theory analysis have attracted tremendous attention in the past decades.54 Wit3Figure Based on the above analysis of ice injury during cryopreservation, it is obvious that effective ice control can weaken the effects of ice injury and improve the efficiency of cryopreservation of cells, tissues, and organs. With the rapid development of materials and chemistry, novel materials with the function of ice tuning have been developed and utilized as ice inhibitors in the process of cryopreservation for a long time Figure 2, provi3.1 To this end, CPAs are used as additives to prevent ice injury in cells. The CPA has received great attention since glycerol was first discovered in 1949. Current commercial CPAs are mainly classified into permeating and nonpermeating types, depending on whether they can permeate into cells. As listed in Table As analyzed in the previous section, the chemical composition of the solution plays a crucial role in the process of freezing and thawing for the successful cryopreservation of various biological samples. Although avoiding ice formation is impossible, strengthening the freezing tolerance of the solution and reducing the ice damage to cryopreserved cells can be achieved.61 To Figure  Meanwhile, the special molecular interaction between DMSO and water alters the freezing process and protects cryopreserved samples. To understand the interaction mechanism of DMSO at the molecular level, Zhang et al. conducted a molecular dynamic simulation between DMSO and water, and the results demonstrated that DMSO could form hydrogen bonds, thus increasing the fraction of unfrozen water and strengthening the freezing tolerance. In addition, the number of formed hydrogen bonds gradually increased as the concentration of CPA increased and the temperature decreased, implying that the special interaction limited the diffusion of water molecules in the freezing process. However, the permeating CPAs probably show toxicity, and even have a side effect on the genes of cryopreserved cells at excess concentration, which restricts DMSO's widespread use in clinical applications. Compared with traditional penetrating CPAs, nonreducing sugars, such as nontoxic and biocompatible trehalose and sucrose, possess hydration and are utilized as ice‐inhibition materials. Recently, it was discovered that sugar can regulate the shape and delay the growth of ice crystals. Additionally, sugars also showed ice‐recrystallization inhibition properties, thus promoting more efficient cryopreservation. Currently, in order to solve the contradiction between the toxicity of the permeating CPAs and cryopreserved efficiency, the combination of permeating CPAs and nonpermeating sugar is an effective strategy to decrease the concentration of permeating CPAs and reduce their toxicity, enhancing cryopreserved cells and tissues.It is widely believed that CPAs have three functions to achieve ice inhibition and promote cryopreservation: ice‐recrystallization inhibition (IRI), freezing point depression, and ice shape and growth control, as presented in Figure 63 Mea3.2 The protective proteins, called AFPs, have unique ice‐crystal control functions. The first ice‐binding AFP was reported in the late 1960s and immediately attracted scientific interest, as it can prevent cryoinjury upon exposure to cold temperature conditions. To date, various AFPs have been found in the body of natural fish, insects, bacteria, etc. As illustrated in Figure  Generally, AFPs reveal three key macroscopic ice‐tuning properties: dynamic ice shaping (DIS), thermal hysteresis (TH), and IRI. The unique ice‐modifying functions of AFPs with respect to DIS and TH are research hotspots and attract significant scientific attention. The underlying mechanism of regulating special ice morphologies for AFPs was ascribed to their absorption‐inhibition effect. AFPs are capable of binding onto the basal or prism plane of ice crystals and lowering ice growth rates, thus shaping the ice, as shown in Figure  The elevated melting point and the depressed freezing point result in temperature hysteresis, which can be explained by the curved and flat ice crystals, according to thermodynamic principles. Interestingly, the hyperactive AFPs possess larger TH gaps, meaning that TH can assess the ice‐tuning capability of AFPs. Ice recrystallization is a process in which smaller ice crystals gradually grow into larger ones at high subzero temperatures. It is a thermodynamically driven process that leads to a decrease in the free energy of the overall system. It was proved that the occurrence of ice recrystallization resulted in dehydration and structural and functional damage to the surrounding tissues of organisms. As schematically illustrated in Figure  Consequently, the preferential binding between the AFPs plane and ice crystal leads to microcurvatures of the ice surface, thus suppressing the growth of the ice crystals. Molecularly, the hydroxyl groups on AFPs can form hydrogen bonds with ice, thus driving the binding effect of AFPs. The solution exhibits decreased ice‐grain area after adding trace amounts of AFPs.Organisms that live in extremely cold environments possess a special cold‐adaptation ability, producing a type of protein to help them survive.71 The AFPs possess two faces: an ice‐binding face (IBF) and a nonice‐bind‐face (NIBF), as revealed in Figure  First, the hyperactive AFP obtained from a beetle was selectively absorbed on a solid substrate. Briefly, the IBF was absorbed by polydopamine and the NIBF was exposed. Furthermore, the NIBF can be bound by (3‐glycidoxypropyl) methyldimethoxysilane (GOPTS), as shown in Figure Another topic, on the effects of AFPs on ice nucleation, is interesting, although many research reports about the influence of AFPs on ice nucleation formation remain debatable.84 AFP The first application of AFPs for cryopreservation began in 1990; Rubinsky et al. discovered that the addition of AFPs could increase the cold‐tolerance of porcine oocytes at hypothermic temperature. Since then, various AFPs have been used in cryopreservation. In another experiment, Lee et al. attempted to apply type III AFPs in the vitrification cryopreservation of mouse ovaries, and the results revealed that the ovaries treated with AFPs possessed higher intact‐follicle ratios than in the control group, implying the positive effect of AFPs in vitrification cryopreservation. Until now, the main cryoprotective mechanism of AFPs in cryopreservation is ascribed to their IRI ability and interaction with cell membranes. We can conclude that AFPs reduce the extracellular ice damage and protect the integrity of cell membranes in freeze–thaw cycles. More recently, to explore the intracellular role of AFPs, Gibson and co‐workers delivered type III AFP into the intracellular location. They found that the excess AFP can cause toxic effects to cryopreserved samples and even lead to gene variation, thereby limiting their widespread applications in the field of cryopreservation. With the rapid development of structural biology, a deep understanding of the correlation between the 3D structure and functions of AFPs will elucidate their ice‐regulation mechanism and facilitate their practical application in the field of cryopreservation.Based on the above‐mentioned unique ice‐control properties, AFPs have become a promising ice‐inhibition candidate for use in cryopreservation.86 The3.3 In particular, for needle ice, the curvature of ice crystals cannot be influenced without antifreeze glycoproteins (AFGPs), and finally, the shape of round flat ice crystals is generated. Once the AFGPs absorb onto the surface of a particular plane of ice, the special surface curvature will increase, resulting in vapor pressure, a subsequent decrease in the melting point, and water molecules being unable to assimilate into surface pockets on the ice. The AFGPs absorption onto the fast‐growing prism facilitates ice growth from the basal plane direction of the ice, producing needle‐shaped ice crystals. It is self‐evident that the needle‐shaped ice crystals can lead to serious mechanical injury for cryopreserved samples when used during cryopreservation. Therefore, it is crucial to develop an artificial synthetic polymer with an ice‐tuning function to overcome the drawbacks of the AFGPs. Currently, with the rapid developments in polymer chemistry, significant progress has been made in designing and synthesizing complex architectures and functional groups, providing a broad platform for achieving AFP‐like polymers but with scalable and stable ice‐modulation functions. Recently, variable small molecular and macromolecular polymers have been introduced into the emerging field of ice inhibition, and thus, have great potential for cellular cryopreservation. Herein, we aim to focus on synthetic anti‐icing polymer materials, including small molecules, ice binding, and amphiphilicity polymers.While AFPs show unique modulation and modifying functions for ice crystals, they are not always appropriate for cryopreservation, owing to drawbacks such as high cost, potential immunogenicity and toxicity effects, large‐scale production, and needle‐shaped ice crystals.40 In  Ben and co‐workers first reported different small‐molecule AFGP analogs as ice inhibitors in 2003; the correlation between carbohydrate hydration and IRI was assessed. They found that the active carbohydrate moiety on C‐linked antifreeze glycoprotein analogs contributed to their IRI ability  onto a gold nanoparticle surface, as shown in Figure  Generally, the atomic distance between the OH groups of PVA is essential for the structure to match with water and ice molecules. In this respect, Gibson and co‐workers showed that the distance between hydroxyls on PVA (2.92 Å) can precisely match with the prismatic plane of the ice crystals (2.74 Å), as illustrated in Figure  Briefly, PVA can suppress the extracellular ice and reduce the ice‐recrystallization damage to cells in the thaw process, as illustrated in Figure  In addition to red blood cells, they also utilized PVA against ice injury and realized glycerol‐free cryopreservation for proteins. Furthermore, it has been reported that PVA can inhibit ice nucleation and facilitate vitrification cryopreservation. In summary, as a well‐recognized ice inhibitor, the IRI of PVA polymer and its further optimization will have positive effects on the cryopreservation field.In addition to small molecules, synthetic macromolecule polymers can be assembled from vast monomers, thereby endowing the polymers with flexible structures and functions. At present, as a typical ice inhibitor, polyvinyl alcohol (PVA) exhibits great potential for ice growth inhibition, and has been utilized for IRI despite its molecular structure differing from that of native AFP. Knight et al. first discovered that the IRI property of PVA that did not show TH performance.101 Si For example, Graham et al. designed a type of facial glycopolymer that contained separate hydrophobic and hydrophilic faces. In order to increase the hydrophobicity, some comonomers were incorporated into the polymer, increasing their solubility and IRI activity  for more successful cryopreservation of oocytes (99.11%) and reduced the CPA concentration from 4.3 to 2.5 m. More importantly, the mitochondrial function of oocytes postcryopreservation was also improved, indicating that the addition of l‐Pro8 can decrease the extracellular ice injury during the freeze–thaw process. The proposed mechanism was attributed to the unique ice growth control of proline, as illustrated in Figure Mimicking the 3D structure of an AFGP offers another strategy to attain ice inhibitors. For native AFP or AFGP, it has been shown that the separated hydrophobic/hydrophilic groups are also a crucial factor for effective ice inhibition. Precious modulation and design of the hydrophobic/hydrophilic domains are essential. Recent research has made remarkable progress in molecular modeling and experiments to elucidate the structure–property relationship of synthetic polymers with ice‐modifying functions.111 Foe Figure . As demo Based on this effect, the influence of polyampholytes on IRI was studied. The underlying mechanism may be that the charge‐balanced nature feature of polyampholytes probably disturbs the quasi‐like water between the water and ice interface, inhibiting the growth of ice crystals. In addition to IRI, it has been found that polyampholytes can be used to inhibit ice nucleation. By regulating the structure of polyampholytes, carboxylated polyampholytes and polyampholytes with carboxyl and amino groups were synthesized. As shown in Figure  At the same time, polyampholytes with carboxyl and amino groups can delay the freezing time from 400 ± 150 s to 7800 ± 2600 s when the temperature decreases from −12 to −20 °C , which shows a similar structure to that of natural AFP; that is, the distance between each monomer is almost the same as that of the GO lattice, which offered a suitable reason for the ice‐inhibition capability of GO.122 Ge The hydrogen bond formation between OQCNs and ice is significant for the ice‐control functions. Interestingly, the OCNs are not capable of exhibiting ice regulation, which can be explained by the difference in atomic arrangement between OCNs and OQCNs, as shown in Figure In addition to GO, different types of oxide quasi‐carbon nitride quantum dots (OQCNs) also showed TH, ice shaping, and IRI effects.70 The As illustrated in Figure Hydrogen bonding interactions play an important role in ice modulation. By designing the surface functional groups to regulate the surface chemistry specificity of nanomaterials, it is feasible to achieve a precise hydrogen bonding taht can match with ice‐crystal planes. In a more recent study, zirconium (Zr)‐based metal–organic framework (MOF) nanoparticles were used to suppress ice recrystallization and melt ice crystals.128 As3.5 In terms of the interactions between monomers that compose the networks, they can be divided into chemical crosslinking and physical crosslinking methods. Chemical crosslinking is mainly based on covalent bonds, and physical crosslinking relies on weaker physical forces such as hydrogen bonds, π‐stacking interactions, and van der Waals interactions. The distinct network structure and composition endow the hydrogel with outstanding biocompatibility, thermal reversibility, optical clarity, and tunable mechanical properties. Recently, anti‐icing hydrogels have been widely reported. The state of the hydrogel and its chemical composition play an important role in the ice‐inhibition performance of hydrogels. In general, the water in a hydrogel can be classified into three states: free water, weakly bound water , and strongly bound water. The strong bound water can maintain its fluidity and remain in a liquid state even when the temperature is below −100 °C, which provides guidance for the design principles of the chemical composition of the hydrogel with ice‐inhibition ability. By introducing ions and alcohol into the hydrogel and modifying its network, the hydrogel can be endowed with ice‐inhibition properties. The underlying mechanisms of hydrogels are mainly attributed to strong interactions such as hydrogen bonds between water molecules and the hydrogel network, thus reducing the fraction of free water. Based on this, a hydrogel material with an ice‐suppression function due to its special chemical composition and state has been widely investigated and applied to cell cryopreservation.Recently, a range of hydrogel materials with ice modulation and inhibition functions have been widely researched and utilized for cryopreservation. Hydrogels are commonly semisolid phases with a network structure.130 In As schematically illustrated in Figure N‐vinylpyrrolidone) (PVPON) based on the layer‐by‐layer (LBL) approach. Then, the PMAA layer was crosslinked with ethylenediamine (EDA) by sacrificial template strategy. Based on the counterion effects, the PMAA hydrogel displayed an 11 °C ice nucleation temperature window and delayed four orders of magnitude of ice‐crystal propagation time, as depicted in Figure  designing hydrophobic polydimethylsiloxane (PDMS) chains with a polyelectrolyte hydrogel  were remarkably higher than without hydrogel (≈20%). Meanwhile, the functional evaluation of recovered stem cells, including stemness and multilineage differentiation, was carried out. The results indicated that the alginate hydrogel used for cryopreservation had no influence on stem cells compared with a fresh group, as exhibited in Figure Unlike other ice‐inhibition materials, the hydrogel can offer an excellent physiological environment for cells, and can be regarded as a cell reservoir owing to its sufficient amount of liquid and bionic crosslinked network structure.141 Ta4Various flourishing engineering strategies offer new paths to inhibit ice and facilitate the efficiency of cryopreservation. In the next section, the promoted cryopreservation based on state‐of‐art ice‐inhibition strategies will be analyzed from the perspective of trehalose delivery, cell encapsulation, and bioinspired structure design.4.1 Currently, the common permeating CPAs such as DMSO can inhibit cryoinjury by hydration and modulate ice formation and growth. However, DMSO is toxic to cells and can cause protein degeneration and gene variation. Moreover, permeating CPAs are difficult to remove from cells, indicating the significance of using nontoxic and new‐type ice‐inhibition materials for weakening, and even avoiding, intracellular ice injury. Trehalose, a biocompatible sugar present in nature, has been utilized as an alternative CPA. Trehalose can protect cells from cryoinjury two ways: 1) enhancing hydration and forming hydrogen bonds, inhibiting ice formation, and 2) forming a glass matrix to restrict metabolic activity. However, trehalose cannot penetrate cell membranes and, thus, acts as an extracellular ice‐inhibition agent. Recently, a series of strategies have been explored to deliver trehalose into cells to reduce intracellular ice damage and improve cryopreservation efficiency. These trehalose‐delivery methods have been reviewed in previous studies. In the next section, we will focus on the novel nanoparticle‐mediated trehalose delivery strategy, and provide several of the latest examples.Intracellular ice formation is a major factor for cell death in the process of cryopreservation.47 Cur Hence, the previous research offers a referential strategy for the delivery of trehalose into cells. In brief, the nanoparticle‐mediated method can induce interactions with the cell membrane and promote membrane permeability. For example, Stefanic et al. designed colloidal bioinspired apatite nanoparticles and promoted trehalose delivery into red blood cells. As shown in Figure  As schematically illustrated in Figure Nanomaterials have been used as vehicles for the delivery of drugs in the medical field, especially for the release of small molecules for targeted therapy of cancer cells.157 HeFigure  After the cellular uptake of nanoparticles containing trehalose, trehalose can be released at pH 5 owing to the acid condition of late endosomes and lysosomes in mammalian cells. Based on this method, the cryopreservation efficiency after adding the pH‐responsive nanoparticles showed a comparable effect to DMSO  from hydrophobic to hydrophilic. As depicted in Figure β cells. As schematically illustrated in Figure β cells, but also the thawed cells still retained blood glucose regulation function and could be transplanted in vivo for diabetes treatment. After transplantation of the cryopreserved β cells into the muscles of the legs, the blood glucose level of diabetic rats can recover the normal range and maintain for two weeks, indicating the functional integrity of β cells after undergoing cryopreservation. In brief, flourishing trehalose‐delivery biotechnology can reduce intracellular ice injury and offers a reliable approach for organic‐solvent‐free cryopreservation.Responsive nanoparticles provide faster and more efficient approaches for cellular trehalose uptake. For instance, as depicted in Figure 153 AfO Figure , demonst4.2 Cell encapsulation methods induce the formation of cell‐laden microcapsules and greatly affect the final cryopreservation efficiency. As shown in Figure  In general, cell encapsulation approaches can be roughly classified into the microfluid, extrusion, and emulsion methods. It is difficult to cover all the cell encapsulation methods in the application of cell cryopreservation; here, we present several typical examples to highlight the role of cell encapsulation in inhibiting ice for cell cryopreservation.Extracellular ice formation and growth is a crucial factor for cell survival in the cryopreservation process. To inhibit extracellular ice and reduce its cryodamage, researchers use biocompatible hydrogels to enclose living cells in capsules, and the protective structure outside the cell can effectively suppress extracellular ice. Especially for the vitrification cryopreservation method, it has been discovered that the cell encapsulation strategy using hydrogel can facilitate the vitrification inside capsules and inhibit the occurrence of devitrification in the process of thawing. More importantly, the 3D microenvironment of the encapsulated hydrogel is similar to the extracellular matrix and allows the diffusion of nutriments and waste products of cells, indicating that the cell‐laden microcapsules, based on encapsulation technology can be regarded as an entirety to cryopreserve, and for further applications such as in vivo transplantation and drug carriers.140 Ce Among extrusion methods, the electrostatic spray technology is typically utilized to produce high‐throughput microcapsules. As shown in Figure  The crosslinked alginate hydrogel network can restrict the movement of water molecules and, thus, inhibit the devitrification. Interestingly, the method can also produce double‐layer microcapsules via tube‐in‐tube devices. The generation of hydrogel microcapsules based on microfluid technology is another typical method. He and co‐workers designed a microfluid device to fabricate cell–alginate hydrogel constructs, as described in Figure  As schematically illustrated in Figure  Moreover, cell encapsulation based on the drop cell printing approach has promising potential for ice inhibition in cryopreservation. Briefly, cell encapsulation is an effective engineering strategy to inhibit the formation and propagation of ice in both cooling and warming procedures, reducing cryoinjury to cells.Extrusion methods are commonly used to fabricate controlled‐sized capsules that effectively suppress devitrification.212 Am4.3In nature, excellent biological organisms are evolving, leading to different morphological and structural functions to adapt and expand in incompatible environments. One of the optimized properties is the unique biological structure that suppresses ice and reduces cryoinjury. For example, the natural cytoderm structure provides a “protective coat” for plant cells, which can avoid the ice crystal directly contacting the organelle and ice nucleation from expanding into the cell. Similarly, some mammalian cells also possess special structural features to protect them from external physical damage. Hence, natural organism structures have offered an important source of inspiration for designing a bioinspired anti‐icing strategy.Figure  Combined with different CPA formulas, the bioinspired core–shell structure showed an excellent protective effect on cryopreserved cells. The mechanism underlying the prevention of ice formation and growth can be described as schematically illustrated in Figure Among various mammalian cells, the zona pellucida structure of egg cells provides a natural “protective coat” for them. Inspired by the zona pellucida structure, core–shell structure cell–biomaterial constructs were designed as displayed in Figure 147 Co Therefore, nanocoating provides a promising method for developing artificial cytoderm structures. As shown in Figure  During the cryopreservation process, the nanocoating not only modulated the interfacial energy between the ice and cell membrane but also resisted the ice propagation into the cell and stopped the permeation of small ice crystals  the physical field can provide sufficient warming rates, thereby inhibiting the occurrence of recrystallization/devitrification and 2) the external applied physical field is able to influence the movement state of water molecules and further modify the formation and growth of ice in the process of cooling. As briefly summarized in Table 173 In5.1 The external magnetic field can affect the movement state of water molecules by orienting, vibrating, and spinning, thereby inhibiting the cluster to some extent and promoting supercooling  based on the magnetic field. As shown in Figure Currently, magnetic freezing technology has been proven to prevent cell destruction by regulating ice crystals and the supercooling degree of the solution.174 Th If the warming rate is not rapid enough, the occurrence of recrystallization/devitrification will cause fatal cryodamage to cells. Hence, the key point is how to achieve fast and uniform recovery during the thawing process. Magnetic iron‐oxide nanoparticles such as Fe3O4 can rapidly convert external magnetic energy into thermal energy, which offers an effective solution to solve the ice‐recrystallization problem. Although the proposed concept of magnetic thawing is relatively recent, as shown in Figure In addition to the freezing process, the external magnetic field remarkably enhanced the thawing efficiency for the recovery of cryogenic cells. It is well known that the warming rate is a significant factor in the survival of cryopreserved biological samples.25 If  Compared with untreated cells, the viability of thawed cells can be remarkably increased. In a later study, they successfully combined magnetic warming with hydrogel encapsulation, as illustrated in Figure  The underlying mechanism of IRI in the warming process was investigated. As depicted in Figure  During the thawing procedure, the magnetic field was capable of providing rapid and uniform warming for the large‐volume PTFE cell container  to inhibit ice growth and enhance the warming rates. As shown in Figure 2 nanosheets and cell samples was directly embedded into liquid nitrogen; subsequently, the laser generated sufficient heating effects to warm the biological samples. Ultimately, the minimum concentration of 0.02% GO improved the final cryopreservation efficiency. Notably, the distribution of GO and MoS2 in the solution is crucial to melt ice crystals uniformly, and to determine the homogeneity of thawing. Hence, it is important to avoid the aggregation of nanomaterials. More recently, Rao and co‐workers proposed laser‐triggered warming to suppress vitrification using soft liquid metal nanoparticles. The nanoparticles showed a higher photothermal conversion efficiency of up to 52%, thus achieving a faster warming rate to reduce ice cryoinjury  on solution crystallization. They discovered that the occurrence of microwave radiation can apparently reduce the fraction of ice crystallization under the same CPA concentration. As shown in Figure m CPA and decreased ice formation by 66% in 4.5 m CPA. Hence, microwave radiation can reduce extracellular and intracellular ice damage during the freezing process and may be a potential method to promote cryopreservation efficiency and quality. In addition to the freezing process, microwaves with higher intensity also play an important role in suppressing devitrification and recrystallization during warming, and have been used for cryopreservation in the rewarming process since the 1970s. When the liquid water absorbs the microwave energy, which results in the occurrence of water molecule friction and increases the temperature of the system. Figure Based on this physical and noncontact ice‐regulation strategy, microwaves have been used for assisting biological cryopreservation. In 1992, Hanyu et al. first applied microwaves for the cryopreservation of small tissue blocks, and described the back mechanism of increasing cryopreservation quality.198 Afe glycol PA on sol5.4−2) has been used to assist freezing and control ice crystals. Similar to other physical field approaches, ultrasound radiation is able to reduce ice size during freezing and enhance heat and mass transfer for cryogenic media, which is important for cell cryopreservation.The propagation of sound waves can generate various physical and mechanical effects on the process of the formation and growth of ice crystals, which is also applied in tuning ice formation and ice size. Power ultrasound with a low frequency (20–100 kHz) and high intensity (>1 W cmcrystals.177 SiFigure  As shown in Figure  Currently, ultrasound thawing technology has been successfully applied in the field of food. Ultrasound with more power can shorten thawing time and further improve the quality of cryopreserved samples (Figure  This method also has promising potential in cell recovery, and reduces ice‐recrystallization damage. It is worth noting that the appropriate parameters of ultrasound, such as radiation duration, intensity, and frequency, are crucial for regulating ice and biological cells. Excessive ultrasound energy might cause mechanical damage to the cell wall and other cell structures. Generally, the underlying ice‐regulation mechanisms of the ultrasonic field are not fully understood; the application of sound waves in the cryopreservation field is in the early stage, and more fundamental research and efforts are needed to establish the relationship between ice regulation and ultrasound field radiation.As illustrated in Figure 202 Ass Figure .[2056As highlighted in this review, innovative materials and strategies for ice inhibition in cryopreservation have made significant progress. We first discussed the fundamental ice cryodamage mechanisms in the cooling–thawing process of cryopreservation. We also summarized current state‐of‐the‐art chemical ice‐inhibition molecules relying on the basic understanding of ice injury, that is, traditional CPAs, AFPs, synthetic polymers, nanomaterials, and hydrogels. Moreover, advanced engineering technologies for ice inhibition, including trehalose delivery, cell encapsulation, and bioinspired structure design, have been emphasized. Ultimately, the control of the formation and growth control of ice crystals based on external physical field strategies was also systematically described. Overall, regardless of intracellular and extracellular ice inhibition, it is vital to achieve high‐efficiency cryopreservation to meet the urgent need for emerging fundamental scientific research and practical clinical applications. Despite these developments, some work remains challenging. First, a deep understanding of the relationships between the 3D structure of AFPs and the ability to tune the formation, growth, and shaping of ice still needs to be fully developed. Second, the universal explanation of natural AFPs for ice inhibition is controversial, and the quantitative analysis of ice inhibition, in theory, has not been established. These challenges will pull scientific researchers to explore a diverse range of bioinspired AFP molecules and/or polymers in structure or functional similarities for ice inhibition and further application in cryopreservation. It should be noted that the availability, cost, and awareness of toxicity are also crucial for assisting the cryostorage of biological samples.With respect to ice‐suppression materials, continuous efforts should be made to explore molecule‐level mechanisms, especially in the molecular interaction between ice‐inhibition materials and water/ice molecules. AFP, as a typical ice inhibitor in nature, provides new inspiration and guidance for anti‐icing materials design. Until now, remarkable progress has been made in the unraveling of the basic mechanism of AFPs on ice nucleation, shaping, and ice recrystallization.40 Des Presently, the duration of organ cryopreservation after procurement greatly decreases the efficiency of organ transplantation and increases cost; therefore, successful cryopreservation of organs is essential for regenerative medicine. In general, cooling and warming rates are crucial factors for organ cryopreservation. Due to the complex extracellular architecture and vascular system, the formation and growth of ice will cause fatal injury to organs, destroying their integrity and function. The most promising method for organ cryopreservation might be vitrification, which avoids ice‐crystal formation and eliminates ice damage during the freezing process. To address this issue, more nontoxic CPAs or other vitrification‐induced materials should be exploited. At the same time, a fast cooling rate is also significant, and can facilitate vitrification. In addition to physical and biological strategies, advanced engineering devices can be developed to accelerate the cooling process. For instance, a recent study reported an ultrarapid cooling approach based on superflash freezing. This method effectively vitrified the cells without adding any CPA, and achieved comparable cryopreservation efficiency to using conventional CPAs. Moreover, the flourishing development of microscopy, magnetic resonance imaging, and microfluid platforms will offer a new understanding of organ cryopreservation and facilitate further development. Another important point is how to inhibit ice recrystallization and devitrification for large‐scale organs during thawing. Uniform and fast space warming based on an external physical field are effective heating methods, especially for magnetic warming. The direction of future development of warming is probably multiphysical and synergetic thawing. For example, the combination of magnetic and laser fields can achieve uniform and fast warming rates, which is essential to overcome the ice barrier. More importantly, the physical field provides both physically and biologically flexible warming, and cannot lead to structure and function variations of cryopreserved samples. In conclusion, with the rapid developments in chemistry, material synthesis, biochemistry, and engineering, we believe that advanced and flourishing cryopreservation will keep pace with current and emerging needs, bringing a bright future for regenerative medicine. We anticipate that this review of previous works will be helpful in promoting the development of safe, high‐quality, and high‐efficiency cryopreservation of biological samples.Advanced engineering strategies such as trehalose delivery, cell encapsulation, and bioinspired structure design have been described as methods to suppress ice and reduce chilling injury. Trehalose delivery can achieve intracellular ice inhibition as the sole nontoxic CPA. Cell encapsulation is capable of providing an integrated platform with ice control, long‐term storage, and cell‐based therapy functions based on cell constructs. Various structures in natural organisms offer a source of inspiration for ice suppression. Despite substantial progress, the main limitation of these methods is the low throughput at the laboratory scale. One of the future directions for investigation should focus on how to achieve macropreservation through these strategies to meet current clinic needs. Moreover, it is clear that cryopreservation single‐cell systems has been successful, whereas cryopreservation of entire organs remains a great challenge.206 PrThe authors declare no conflict of interest.


Several milk/whey derived peptides possess high in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. However, in some cases, poor correlation between the in vitro ACE inhibitory activity and the in vivo antihypertensive activity has been observed. The aim of this study is to gain insight into the structure-activity relationship of peptide sequences present in whey/milk protein hydrolysates with high ACE inhibitory activity, which could lead to a better understanding and prediction of their in vivo antihypertensive activity. The potential interactions between peptides produced from whey proteins, previously reported as high ACE inhibitors such as IPP, LIVTQ, IIAE, LVYPFP, and human ACE were assessed using a molecular docking approach. The results show that peptides IIAE, LIVTQ, and LVYPFP formed strong H bonds with the amino acids Gln 259, His 331, and Thr 358 in the active site of the human ACE. Interestingly, the same residues were found to form strong hydrogen bonds with the ACE inhibitory drug Sampatrilat. Furthermore, peptides IIAE and LVYPFP interacted with the amino acid residues Gln 259 and His 331, respectively, also in common with other ACE-inhibitory drugs such as Captopril, Lisinopril and Elanapril. Additionally, IIAE interacted with the amino acid residue Asp 140 in common with Lisinopril, and LIVTQ interacted with Ala 332 in common with both Lisinopril and Elanapril. The peptides produced naturally from whey by enzymatic hydrolysis interacted with residues of the human ACE in common with potent ACE-inhibitory drugs which suggests that these natural peptides may be potent ACE inhibitors. The number of people with unhealthy living habits who have developed cardiovascular disease (CVD) has increased in recent years. The WHO reported that an estimated 17.9 million people lose their lives as a result of cardiovascular disease every year . High blACE  is a zinc metallopeptidase, found in male genital, vascular endothelial, neuro-epithelial, and absorptive epithelial cells ,8,9, andConsequently, ACE-inhibiting natural products have been vigorously investigated during the last decades, due to their potential in lowering blood pressure during hypertension. Among various types of bioactive peptides, ACE-inhibitory peptides from food sources have been most extensively studied for their potential use as natural alternatives to drugs for reducing blood pressure through binding and inhibiting ACE, and thus preventing and managing hypertension ,15. FoodAlthough different animal and plant proteins have been used in the development of functional foods providing antihypertensive activity, milk is the main source of antihypertensive ACE-inhibitory peptides reported to date . Milk isThe activity of these peptides depends on their inherent amino acid composition and sequence ,30. ShorACE inhibitors are commonly discovered using classic investigation techniques which include hydrolysis of proteins with different proteolytic enzymes, isolation and purification of peptides using chromatographic systems, and synthesis of corresponding peptides for the confirmation of activity and structure . AlthougHerein we investigate the potential interactions between whey protein derived peptides with high ACE inhibitory activity and human ACE, utilising a molecular docking approach. This study follows our previous work , where tAccording to the EMBOSS NEEDLE results , there iMolecular docking was conducted to elucidate the potential molecular interactions between the whey-protein derived peptide sequences and specific amino acids at the binding site of human ACE. The peptide sequences were docked into the binding site of the human ACE, using the X ray crystallographic structure of the human ACE receptor (PDB code 6F9V). The extracted co-crystallized ligand, Sampatrilat,  was firsHydrogen bonds are a significant factor that contribute to the specificity and stability of protein-ligand interactions. Hydrogen bonds interactions were demonstrated to play a crucial role in stabilizing the docked ligand complexes . The disN-{1-[2-carboxy-3-(N-mesyllysylamino) propyl]-1-cyclopentylcarbonyl} tyr-osine) -r-osine)  is a potr-osine) . Overall, the docking results together with comparisons with the ACE inhibitory drugs provide strong evidence for the ACE inhibitory activity of IIAE, LIVTQ, and LVYPFP. In our previous work , IIAE, L50 is defined as the peptide concentration required to reduce the ACE activity by half. In our previous work where we characterized angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins , peptidehttps://www.ebi.ac.uk/) was queried for human and rabbit ACE amino acid sequences, together with known three-dimensional protein structures. Reviewed sequences were selected and the protein sequence files were downloaded. The accession codes for the human ACE and the rabbit ACE used in this work are P12821 and P12822, respectively. The two sequences were then uploaded to Emboss Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle/) for multiple sequence alignment and comparison.EMBL-EBI  , as provWhey protein-derived peptides Ile-Pro-Pro (IPP), Leu-Ile-Val-Thr-Gln (LIVTQ), Ile-Ile-Ala-Glu (IIAE), and Leu-Val-Tyr-Pro-Phe-Pro (LVYPFP) were used as ligands in separate docking runs. Docking was performed using the docking algorithm Surflex-Dock, as provided in Sybyl-X 2.1. The X-ray crystallographic structure of sampatrilat-Asp in complex with Angiotensin-I-converting enzyme  retrieved from the protein data bank (PDB) was chosen as the target protein for the docking studies, based on its high resolution structure co-crystallized with sampatrilat-Asp .The Biopolymer Structure Preparation Tool, with the implemented default settings provided in the SYBYL programme suite, was used to prepare the protein structure for docking; hydrogens were added to the protein structure in idealised geometries, backbone and sidechains were repaired, residues were protonated, sidechain amides and sidechain bumps were fixed, stage minimization was performed, and all water and any ligand molecules were removed. The three-dimensional (3D) structure of each ligand was constructed, using the “Build Protein” tool, as provided in Sybyl-X. Once constructed, charges were assigned to each atom of each molecule, using Merck Molecular Force Field (MMFF94) charges. Localized energy minimizations were then performed, and the final structure for each ligand in its lowest energy conformation was used for subsequent docking experiments. The resulting 3D coordinate files were converted to a MOL2 format for subsequent use in Surflex-Dock experiments, as provided in the SYBYL-X 2.0 software suite.Surflex-Dock is a search algorithm that utilizes an empirically derived scoring function whose parameters are based on protein-ligand complexes of known affinities and structures. This method employs a “protomol”, which is an idealized active site, as a target to generate presumed poses of molecules or molecular fragments. The protomol is employed as a mimic of the ideal interactions made by a perfect ligand to the active site of the protein. This molecular-similarity based alignment allows for optimization of potentially favorable molecular interactions, such as those defined by van der Waals forces and hydrogen bonds. In the present work, the protomol was defined by optimizing the threshold and bloat values to 0.5 and 0, respectively, to create a protomol that adequately described the binding pocket of interest. The extent of the protomol and its degree of coverage of an active site are controlled by these two parameters: the threshold value indicates the amount of buried-ness for the primary volume used to generate the protomol, and the bloat parameter determines the number of Ångstroms by which the search grid beyond that primary volume should be expanded. It is generally better to err on the side of a small protomol than on a protomol that is too large . All parMolecular interactions, for the docking results, are reported according to the Sampatrilat (PDB code 6F9V) amino acid sequence numbering; for comparisons between different sequence numbering in studies referred here See ). The soFor the first time, reported herein, potential interactions between the naturally produced peptides from whey and ACE have been investigated, using a molecular docking approach. Peptides, IPP, IIAE, LIVTQ, and LVYPFP formed strong H bonds and salt bridge interactions with residues in the active site of human ACE. Moreover, a comparison with commercial ACE inhibitory drugs showed that the natural peptides interacted similarly to the drugs mimicking the same interactions with ACE active site residues. This study provides strong evidence for the ACE inhibitory activity of milk derived peptides, which have not been tested in vivo before. The results of this study, of novel milk derived whey peptides, could lead to the production of novel ACE inhibitors.


Background: Gianotti Crosti syndrome (GCS) is a self-healing condition with a spontaneous resolution in 2–6 weeks but, even if rarely, recurrent episodes have been reported.Objective: The aim of this observational study is to investigate serum Immunoglobulin E (IgE) level in children with GCS, evaluating if there is a relationship between IgE level and clinical course of the disease.Methods: Children with GCS diagnosed at a tertiary care children's university hospital between June 2018 and November 2019 were prospectively enrolled. Demographic, clinical and hematochemical data of children investigated were collected. In particular, IgE level were investigated at symptoms onset and, if available, at the following blood tests. Patients were divided in 2 groups on the bases of the clinical course: children with a chronic relapsing course and children who did not present any relapse.Results: Among 29 patients enrolled in this study, 14 (48.3%) children had a chronic relapsing course and 15 (51.7%) did not present any relapse. A statistically significant difference was present considering the length of the disease: 210 days (IQR: 161.25–255) for patients with a chronic relapsing course compared to 40 days (IQR: 30–75) for the other group (p < 0.0001). About the median IgE level in the 2 groups, a value about 10 time higher was found in children with chronic course compared to the other group  with a statistically significant difference (p < 0.0001).Conclusion: Despite the study limitations, a significant correlation between higher IgE levels and chronic-relapsing course of the GCS can be assumed. Gianotti-Crosti syndrome (GCS), or infantile papular acrodermatitis, is a distinctive childhood exanthema characterized by papular or papulovescicular lesions that are most prominent on the extremities, buttocks and face , 2. AlthIt is usually a self-healing condition with a spontaneous resolution in 2–6 weeks, but sometimes it may last several months and recurrent episodes have been reported. No specific treatment for GCS is required, except mild topical steroid cream and oral antihistaminic which may be prescribed for the itch. Recurrence of GCS has been reported in literature , 7. AccoThe aim of this observational study is to investigate serum Immunoglobulin E (IgE) level in children with GCS and its relationship between the IgE level and the clinical course of the disease. Moreover, IgE level within the first month of disease would be assessed in order to establish a possible predictive role on the length of GCS.All the children with a diagnosis of GCS at the Anna Meyer tertiary care children's university hospital were prospectively enrolled between June 2018 and November 2019. Demographic, clinical and hematochemical data were collected. In particular, the following data were included in the study database: children's sex and age, date of symptoms onset, clinical prodromes before GCS onset, personal history of atopy, disease length, and blood tests result. IgE level were investigated at the first hospital evaluation and, if available, at the following blood tests during follow-up visits.According to local ethical review board instructions, on hospital admission all patients signed an informed consent for the inclusion in observational studies, with anonymized data extraction.GCS was clinically diagnosed through validated diagnostic criteria for GCS proposed by Chuh et al. , Chuh 1. In part2 test or Fisher test, as appropriate. Quantitative variables were reported as median and interquartile ranges (IQRs) and they were compared by means of nonparametric tests (Mann–Whitney U). P < 0.05 was considered statistically significant. Pearson's correlation coefficient (r) has been calculated to see the correlation between IgE levels and time of the blood tests. To assess the diagnostic accuracy of serum IgE levels to predict the chronic relapsing course risk of GCS, we calculated sensitivity, specificity, and the area under the receiver operating characteristic (ROC) curve (AUC).Data were recorded in the study database and analyzed by SPSS Statistics, 24.0 statistics software. Categorical variables and frequencies were compared by means of χn = 12, 44.8%), in particular mild to moderate fever, cough, diarrhea, and vomiting or influenza-like symptoms. One patient reported the onset of the lesions 5 days after the immunization for rotavirus.A total of 29 patients with GCS  were enrolled during the study period. The median age at onset was 20 months (IQR: 13–29) with a median length of disease of 90 days (IQR: 40–210). Infective prodromes were present in almost half of the patients . On the contrary, a statistically significant difference was present considering the length of the disease: 210 days (IQR: 161.25–255) for patients with a chronic relapsing course compared to 40 days (IQR: 30–75) for the other group (p < 0.0001). The median time between symptoms onset and the first IgE detection was 17 days (IQR: 3.5–50). For children with a chronically relapsing course this interval was in median of 30 days (IQR: 11–98.75), compared to the other group of 10 days (IQR: 1–20) (p < 0.037). Children with a chronically relapsing course displayed also extensive and more itchy lesions than the other group.Patients were divided in 2 groups on the bases of the clinical course: 14 (48.3%) children had a chronic relapsing course and 15 (51.7%) did not present any relapse, as shown in r = 0.356, p = 0.005). IgE levels within individual patients are reported in p < 0.0001). Moreover, the 2 groups were compared considering only the patients for whom IgE value was available within the first month since disease start. Therefore, this analysis was limited to a subgroup of 21/29 patients. IgE level was significantly higher in patients with a successive chronic course with respect to the patients with a shorter course. In particular, median IgE levels were 1,144.5 U/mL  and 93 U/mL (IQR: 39.5–259), respectively (p = 0.003). A very high value of IgE  was detected in 1 patient with a chronic relapsing course of GCS. The clinical and laboratory scoring system for HyperIgE syndrome (HIES) .Available IgE levels during the disease course are showed in e (HIES)  was applPersonal history of atopy or allergopathy was negative in the study patients, with the exception of 1 child who was positive for specific IgE for food. A complete allergy screening was not performed in the enrolled children, as no children presented anamnestic and clinical criteria suggestive for atopy diathesis . No data about family history of atopy was available.p = 0.121). In particular, mild eosinophilia  was found in 11/14 patients (78.6%) and 6/11 (54.5%) didn't present a chronic course of GCS. Severe eosinophilia  was found in 21.4% (3/14) of the children, and one of them didn't present a chronically relapsing course  had a chronically relapsing course. This group of patients had a duration of the disease 5 times longer than the other group. If we compared the median IgE level between the 2 groups, we found a 10-fold IgE value in children with chronic course. Furthermore, we found that this difference of serum IgE value, was already present if the blood tests were done within the first month since onset of the disease.STAT 3 of hyper IgE syndrome. A similar case was also described by Chuh  who was confirmed with the pathogenic variant in  by Chuh . MoreoveIn order to investigate other possible markers of GCS with chronic relapsing course, in this study we analyzed also AEC. This was done considering that in other conditions, such as allergies, IgE levels and AEC are both increased. In fact, as GCS is thought in part to be related to an immunologic response to a viral infection, potentially of hypersensitivity, eosinophilia or high IgE may result. The AEC, detected within 2 months since the diagnosis of GCS, was available for 21/29 patients and no statistical significance was found comparing the 2 groups. This led us to hypothesize that AEC has not a direct correlation with the GCS course. The lack of correlation with blood eosinophilia is not surprising in the light of a histopathological finding of GCS characterized by the presence of an inflammatory infiltrate mainly of lymphocytes and histiocytes with occasional scattered eosinophils , 15. Desvs. 6.8% and 51.7 vs. 31%, respectively)  has been reported and, not rarely, GCS has been described following vaccination –6. This ctively) . LikewisHowever, in our study a complete allergic screening was not performed for the absence of suggestive clinical symptoms. Differently from those studies we didn't found a correlation with personal history of atopy and with related manifestations, so we cannot confirm the previously reported data. The limited follow-up does not consent to exclude that these children may develop atopic dermatitis or other allergic symptoms in the future, since we followed them in a short time period. Moreover, IgE levels after symptoms resolution were available only for 12/29 patients, and therefore were not analyzed in this study. This was due to the fact that many patients did not come back to visit after clinical recovery. Therefore, it is not possible to assume that there was a complete normalization of IgE levels in both groups and, on the other hand, to define if there was a persistence of this condition.Otherwise, we focused on the possible role of IgE in the pathogenesis or in the clinical evolution of GCS. The main result of our study is that the higher value of IgE, when detected within the first month to symptoms onset, is a direct predictive factor of the course of GCS. This is important because, even if GCS is a transient and self-healing condition, chronically relapsing course is very common and often associated with itchy symptoms. The knowledge of a possible long-lasting disease supports the physician in reassuring children's parents.The main limitation is that study group was small and may not be representative of the entire population of children with GCS. Moreover, another limit is the not homogeneous time of blood testing. In fact, despite the prospective design, blood tests were done only in occasion of clinical evaluation. Therefore, IgE levels were not available at the same time for all patients. No microbiological data, including viral serologies, were available, so it was not possible to establish if underlying superinfection could play a role in the chronicity of the lesions. No complete allergological data were available, in particular allergen specific IgE were not done in absence of a personal history of atopy. Moreover, the lack of IgE data after symptoms resolution did not consent to draw conclusions regarding possible differences on the timespan of this alteration. Therefore, we believe that further prospective studies in children could better clarify the role of IgE levels and the immune background of this condition.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.AB and EV wrote the main manuscript. FP, SR, and TO contributed to the data collection. CF, CA, and LG are the senior supervisors of the work for the dermatologic, immunologic, and pediatric infectious section, respectively. All authors contributed to the article and approved the submitted version.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.


Tetracera scandens) is a Southeast Asian medicinal plant that has been traditionally used for the management of diabetes mellitus. The underlying mechanisms of the antidiabetic activity have not been fully explored yet. Hence, this study aimed to evaluate the α-glucosidase inhibitory potential of the hydromethanolic extracts of T. scandens leaves and to characterize the metabolites responsible for such activity through gas chromatography–mass spectrometry (GC–MS) metabolomics. Crude hydromethanolic extracts of different strengths were prepared and in vitro assayed for α-glucosidase inhibition. GC–MS analysis was further carried out and the mass spectral data were correlated to the corresponding α-glucosidase inhibitory IC50 values via an orthogonal partial least squares (OPLS) model. The 100%, 80%, 60% and 40% methanol extracts displayed potent α-glucosidase inhibitory potentials. Moreover, the established model identified 16 metabolites to be responsible for the α-glucosidase inhibitory activity of T. scandens. The putative α-glucosidase inhibitory metabolites showed moderate to high affinities  upon docking into the active site of Saccharomyces cerevisiae isomaltase. To sum up, an OPLS model was developed as a rapid method to characterize the α-glucosidase inhibitory metabolites existing in the hydromethanolic extracts of T. scandens leaves based on GC–MS metabolite profiling.Stone leaf ( Omics technologies, including genomics, transcriptomics and proteomics, aim to give a holistic view about a biological system by studying different subcellular components  . MetabolClinacanthus nutans chromen-2-one (Saccharomyces cerevisiae) crystal structure, obtained from the Protein Data Bank (PDB ID: 3A4A) using AutoDock Vina. The docking method was initially validated through control docking of the experimentally bound ligand  before proceeding to dock the putative active metabolites , linoleic acid (3), α-linolenic acid (4), 1-monopalmitin (5), catechin (10), α-tocopherol (11), stigmasterol (12), β-sitosterol (13), 1-triacontanol (14), cycloartenol (15) and 24-methylenecycloartenol acetate (16) have disappeared completely from the chromatogram of the water extract. These qualitative and quantitative differences can explain the great difference observed in the α-glucosidase inhibitory activity of both extracts.Two unsaturated fatty acids; linoleic acid (C18:2) and α-linolenic acid (C18:3) as well as two saturated ones; stearic acid (C18:0) and palmitic acid (C16:0) in addition to its glyceryl ester, 1-monopalmitin were identified by the multivariate model as α-glucosidase inhibitors. The α-glucosidase inhibitory activity of the free fatty acids has been proposed previously by similar metabolomics studies  and has Another class of identified α-glucosidase inhibitory metabolites was sterols, represented by stigmasterol, β-sitosterol, cycloartenol and its derivative 24-methylenecycloartenol acetate. The α-glucosidase inhibitory activity has already been reported for stigmasterol and β-sitosterol ,36,37, wCassia obtusifolia seeds and assayed for their α-glucosidase inhibitory potential. Emodin showed high activity with an IC50 value of 1.02 µg/mL, while questin showed much less activity with an IC50 of 136.19 µg/mL chromen-2-one as well as 1-triacontanol were also identified by the multivariate model as α-glucosidase inhibitors. The α-glucosidase inhibitory activity has already been reported to the first 3 metabolites either theoretically via similar metabolomics studies or experimentally via an in vitro enzyme assay chromen-2-one and 1-triacontanol. Molecular docking results showed that α-tocopherol, 5-methoxy-8,8-dimethyl-10-(3-methyl-2-butenyl)-2H,8H-pyranochromen-2-one and catechin exhibit high affinity towards the active pocket of the enzyme as indicated by their low binding energies , whereas phytol showed moderate affinity . By investigating the interactions in the best docked conformation–enzyme complexes for these metabolites, it was noticeable that both catechin and 5-methoxy-8,8-dimethyl-10-(3-methyl-2-butenyl)-2H,8H-pyranochromen-2-one formed only a single hydrogen bond  compared to 2 hydrogen bonds for α-tocopherol (with Arg315 and Asp307) and phytol (with Arg446 and Asp352).Anthraquinones  is a widely distributed class of phenolic plant secondary metabolites. Natural as well as synthetic anthraquinones are known for their laxative, antioxidant, antibacterial, antifungal, antiviral, anti-inflammatory and anticancer activities ,39. Many19 µg/mL . The molme assay ,44. To oTetracera scandens is a traditional antidiabetic herb, widely distributing in Southeast Asian countries. This study demonstrated the significant α-glucosidase inhibitory potential of the methanolic extracts of T. scandens leaves. Moreover, an OPLS multivariate model was developed to correlate the mass spectral data of the prepared extracts to the corresponding α-glucosidase inhibitory IC50 values. GC–MS based profiling of the active metabolites led to the characterization of 16 metabolites as α-glucosidase inhibitory compounds, viz.: palmitic acid, phytol, linoleic acid, α-linolenic acid, 1-monopalmitin, 5-methoxy-8,8-dimethyl-10-(3-methyl-2-butenyl)-2H,8H-pyranochromen-2-one, stearic acid, questin, emodin, catechin, α-tocopherol, stigmasterol, β-sitosterol, 1-triacontanol, cycloartenol and 24-methylenecycloartenol acetate. Furthermore, a molecular docking study was carried out to predict the binding affinities and the possible interactions of the ligand–enzyme complexes. The putative α-glucosidase inhibitory metabolites showed moderate to high affinities toward the active pocket of Saccharomyces cerevisiae isomaltase as indicated by their predicted binding energies . Conclusively, this study demonstrated the α-glucosidase inhibitory potential of the hydromethanolic extracts of T. scandens leaves and determined the metabolites that elicited this activity through the GC–MS-based metabolite profiling approach.


VGLUT2 mRNA, but cortical neurons do not. These observations support our theory that the subcompartments revealed by CO histochemistry represent the distribution of thalamo-cortical afferent terminals in the primate visual cortex.Cytochrome oxidase (CO) histochemistry has been used to reveal the cytoarchitecture of the primate brain, including blobs/puffs/patches in the striate cortex (V1), and thick, thin and pale stripes in the middle layer of the secondary visual cortex (V2). It has been suggested that CO activity is coupled with the spiking activity of neurons, implying that neurons in these CO-rich subcompartments are more active than surrounding regions. However, we have discussed possibility that CO histochemistry represents the distribution of thalamo-cortical afferent terminals that generally use vesicular glutamate transporter 2 (VGLUT2) as their main glutamate transporter, and not the activity of cortical neurons. In this study, we systematically compared the labeling patterns observed between CO histochemistry and immunohistochemistry (IHC) for VGLUT2 from the system to microarchitecture levels in the visual cortex of squirrel monkeys. The two staining patterns bore striking similarities at all levels of the visual cortex, including the honeycomb structure of V1 layer 3Bβ (Brodmann's layer 4A), the patchy architecture in the deep layers of V1, the superficial blobs of V1, and the V2 stripes. The microarchitecture was more evident in VGLUT2 IHC, as expected. VGLUT2 protein expression that produced specific IHC labeling is thought to originate from the thalamus since the lateral geniculate nucleus (LGN) and the pulvinar complex both show high expression levels of  The primate visual system is subdivided into multiple parallel pathways, and this segregation appears as laminar and columnar domains in the visual cortex  were used in this study. Among them, four hemispheres from three monkeys were used for cortical tangential staining: The right cortex of ID 18–20, male, 1,100 g, that was subjected to intravitreous injection of tetrodotoxin  into the right eye 1 day before perfusion, the right and left cortices of ID 18–27, female, 500 g, that was subjected to intravitreous injection of TTX (1 mM−5 μL) into the left eye 2 days before perfusion, and the right cortex of ID 18–30, male, 1,000 g, that was subjected to left eye enucleation 14 days before perfusion. All procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University, and were in accordance with the guidelines of the National Institutes of Health (NIH), United States of America.The brains of three male and three female adult squirrel monkeys ], followed by 0.5–4.0% paraformaldehyde (PFA) in 0.1M PB. The brain was removed from the skull, and the visual cortex was separated from the rest of the brain and flattened immediately. The flattened visual cortices were immersed in a post-fixative (30% sucrose/4% PFA in PB) at 4°C overnight and cut tangentially at 40 μm using a freezing microtome. The remaining brain tissue was immersed in 30% sucrose in PB at 4°C for ~1 week until the tissue sank to the bottom of the container. Then, the brain tissue was cut coronally at 40 μm using a freezing microtome. Sections were stored at −20°C in a cryoprotectant solution  until used.The animals were given an overdose of sodium pentobarbital (>50 mg/kg body weight) and were perfused with a sucrose solution , whereas that outside CO blob was 189.25 ± 22.3 per mm2 (counted for 318 ROIs of 0.2 × 0.2 mm square from 78 interblobs in 4 hemispheres of 3 monkeys), and there was no statistical significance between them . This result suggested that CO-rich cortical neurons did not constitute a major component of the CO blobs. CO intensity was higher overall in the blobs compared to the areas surrounding the blobs, but the detailed structure was unclear in the CO histochemistry  has been confirmed in several previous studies  and intercalated pale stripes were discernable throughout V2 in tangential CO stained sections , as repo pattern . This re2 (counted for 92 ROIs of 0.2 × 0.2 mm square from 20 CO dark stripes in 4 hemispheres of 3 monkeys), whereas that outside CO dark stripes was 249 ± 9.0 per mm2 , and there was no statistical significance between them . This result implied that the presence of CO-rich cortical cells did not cause the pattern of thick and thin stripes. On the other hand, the VGLUT2-ir neuropil and puncta were abundantly observed in the thick and thin stripes but not in the pale stripes . In the right V2 of ID 18–20, the CO dark stripes/VGLUT2-ir rich stripes was 0.59 and the VGLUT2-ir rich stripes/CO dark stripes was 0.62. Average proportion of the CO dark stripes/VGLUT2 in the right and left V2 of ID 18–27 was 0.69 and the VGLUT2-ir rich stripes/CO dark stripes was 0.66. In the right V2 of ID 18–30, the CO dark stripes/VGLUT2-ir rich stripes was 0.77 and the VGLUT2-ir rich stripes/CO dark stripes was 0.77. Altogether, the ratio of CO dark stripes/VGLUT2-ir rich stripes was 68.3 ± 7.4% and that of VGLUT2-ir rich stripes/CO dark stripes was 68.4 ± 6.5% (n = 3 each). Overall, significant overlap was observed in this analysis as well.The quantification of overlap in V1 blobs and V2 stripes was done with manual drawing of contours. To confirm that the results were not biased by subjectivity of investigators, we used an automated program to quantify the proportion of overlap in selected small regions , whereasVGLUT2 mRNA to study the lateral geniculate nucleus (LGN) and the pulvinar complex. Even though we attempted monocular inactivation using TTX injection (1 mM−3 μL) into the left eye in this specific animal (ID 19-04) 2 days before perfusion, no apparent difference in gene expression or CO staining pattern was observed between the different layers or hemispheres. Therefore, we considered that the histochemical patterns in this subject were similar to those of visually intact animals.To address the possibility that the VGLUT2 protein observed in V1 and V2 was derived from the thalamus, we used ISH for VGLUT2 mRNA signal was observed in all LGN layers (The squirrel monkey LGN consists of magnocellular (M) layers, parvocellular (P) layers, and koniocellular (K) layers, as seen in other primates (Hess and Edwards, N layers . Based oN layers .VGLUT2 mRNA signals were abundantly and uniformly observed in all pulvinar nuclei (VGLUT2 sense probe did not reveal any signal above background (Similar results were observed in the pulvinar. In r nuclei . Neuronar nuclei . In gener nuclei  (Figure r nuclei . The VGLckground .The purpose of this study is to demonstrate how the expression patterns of CO and VGLUT2-ir correspond with each other. The close correspondence between labeling patterns of the two staining methods supported the possibility that CO-rich domains such as V1 blobs and V2 thick and thin stripes in the primate visual cortex are distinct because they contain abundant afferent terminals from the thalamus. VGLUT2 is an excellent endogenous molecule to study thalamo-cortical connectivity patterns because it is strongly expressed in primary neurons of all sensory thalamic nuclei, but not in the cortex. Also, a majority of the VGLUT2 protein is transported and stored in axon terminals (Nahmani and Erisir, VGLUT2 mRNA is robustly expressed in all LGN layers, and VGLUT2-ir is distinctly observed in all V1 layers and domains described above. Although previous studies suggested that there is some projection from the pulvinar to layer 1 of V1 (Moore et al., Visual information of different modalities is processed separately in parallel pathways of the LGN and visual cortex Lund, , as descTraditional tracer studies have revealed that feedforward projections from the pulvinar complex terminate in the middle layers of CO-rich thick and thin stripes in V2 (Livingstone and Hubel, Furthermore, we consider that this principle could be applied to more extrastriate visual cortices beyond V2. The pulvinar complex of primates consists of multiple nuclei, and the PL primarily sends axons into V2 (Ungerleider et al., Finally, the staining patterns were quite different between ISH and IHC for VGLUT2 in the pulvinar . The locWe consider that thalamic neurons generally possess higher metabolic activity than cortical neurons. Therefore, when the cortex is stained using CO histochemistry, clusters of the thalamo-cortical projection terminals may stand out compared to other regions (Takahata, Several electron microscopic studies suggested that CO-reactive mitochondria mainly reside in dendrites rather than axons in squirrel monkeys and macaques (Carroll and Wong-Riley, The similarity between CO staining pattern and thalamo-cortical afferent distribution has been pointed out in some previous studies as well (Livingstone and Hubel, The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Zhejiang University.SY and TT designed research and wrote the manuscript. SY, QZ, SL, and TT conducted experiments. All authors contributed to the article and approved the submitted version.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.


Biomarkers of clinical efficacy for subcutaneous immunotherapy (SCIT) on allergic rhinitis (AR) have not been identified yet. This study aims to assess the clinical relevance of serum inhibitory activity for IgE by the method of enzyme-linked immunosorbent facilitated antigen binding (ELIFAB) during SCIT for Artemisia-sensitized AR patients.19 AR patients were studied who had undergone Artemisia-specific SCIT for more than 8 months . Peripheral bloods were collected before and after treatment. The serum inhibitory activity for IgE was tested by ELIFAB and the level of Artemisia-specific IgG4 (Artemisia-sIgG4) was determined by ELISA. Clinical improvement was evaluated based on the symptom scores and rescue medication use (SMS). The 2-tailed Wilcoxon signed-rank test and the Spearman rank test (two-tailed) were used to analyze data by using SPSS 20.0, with P values of less than 0.05 considered as significant.The SMS decreased significantly after SCIT , the treatment was remarkably effective for 6 patients, effective for 10 and ineffective for 3, along with a total effective rate 84.21%. The serum inhibitory activity for IgE increased significantly after SCIT (P < 0.05) and was correlated with the levels of Artemisia-sIgG4 . The levels of Artemisia-sIgG4 elevated dramatically after treatment (P < 0.01) and were related with the duration of treatment . But there was no relationship between clinical improvements and the serum inhibitory activity for IgE.The serum inhibitory activity for IgE increased significantly after SCIT, however, there was no correlation between it and clinical improvements by statistics analysis. So whether the serum inhibitory activity for IgE can act as biomarker of efficacy for SCIT or not needs to be studied further. Allergic rhinitis (AR) is an inflammatory disease of the nasal mucosa, induced by an IgE-mediated reaction in atopic subjects . In the Recently Artemisia is reported to be the most common outdoor aeroallergen in Beijing  so it’s In this study, Artemisia-sensitized AR patients were chosen as subjects, and the main purpose was to analyze the clinical relevance of the serum inhibitory activity for IgE tested by ELIFAB assay, by detecting the serum before and after subcutaneous immunotherapy (SCIT), one of the predominant forms of AIT. It was a self-controlled study, that is, indicators were compared before and after SCIT for each individual subject.Patients with AR were enrolled in the study who sought treatment at Department of Allergy, Capital Medical University affiliated Beijing Shijitan Hospital between September, 2016 and May, 2018. They should meet the following criteria: (1) be diagnosed with allergic rhinitis according to ARIA 2008 diagnostic criteria, with or without asthma ; (2) hadBefore the initiation of SCIT, blood sample was collected and clinical evaluation was done for each subject. Also, when they came back to the outpatient for follow-up visit after more than 8 months SCIT, blood samples were collected and clinical evaluation were done again.Serum samples from subjects were collected before and after SCIT, immediately centrifuged at 1500 rpm, 10 min, 4 °C, and stored at − 70 °C until used for detection.All the subjects enrolled in the study suffered allergic symptoms, like sneezing, rhinorrhea in late summer and autumn and were diagnosed as seasonal AR. They underwent SPT testing with extracts of four main autumn pollen allergens in Beijing area . The subjects discontinued the antihistamine at least 72 h before SPT testing. The positive control was histamine hydrochloride (10 g/L) and the negative control was glycerine saline. After disinfecting the palmar skin of the subjects’ forearm, one drop of extract fluid, one drop of negative control fluid and one drop of positive control fluid were placed at an interval of 2 cm. The standard needle was inserted into the dermis vertically through the droplet, and was removed vertically after being maintained for 1 s. The result would be determined in 15 min.Wheal diameter = /2. The diameter of the wheal for positive control group should be > 3 mm, while the negative control group should have no wheal. Skin index (SI) = allergen wheal diameter/histamine wheal diameter. Skin reactivity was graded according to SI: 0 < SI < 0.5 was “+”; 0.5 ≤ SI < 1 was “++”; 1 ≤ SI < 2 was “+++”; SI ≥ 2 was “++++”. Skin reactivity to allergens ≥++ was regarded as SPT positive. It’s reported that reactivity ≥++ has the strongest screening ability for Artemisia AR .2, which was the highest concentration of updosing phase, the maintenance phase started. 0.5 ml of extracts was injected twice a week. After maintenance for 1 year, it could be changed to once a week as appropriate. The SCIT protocol was shown in Fig. The patients were treated with s.c. injections of standardized Artemisia allergen extracts . The regular SCIT protocol included updosing phase and maintenance phase. The initial concentrations of extracts were set according to the SPT levels of subjects and the higher the level, the lower the concentration. The patients were injected with 0.1 ml extracts with the initial concentration for the first time. The dose increased by 0.1 ml for each subsequent injection and it became 1 ml for the 10th injection. Then extracts with 10 times higher concentration was used and similar process was repeated. Injections were given twice a week, extracts of each concentration could be used for 5 weeks. When the concentration became 1:10In the study, subjects were treated with multiallergen immunotherapy as most of them were polysensitized to more than one allergen. The types of allergens in SCIT corresponded to patients’ sensitization spectrum. All of them took Artemisia SCIT.The serum levels of Artemisia specific IgE (Artemisia-sIgE) were determined in Department of Allergy, Capital Medical University affiliated Beijing Shijitan Hospital.The concentrations of Artemisia specific IgG4 (Artemisia-sIgG4) in sera were measured by a four-layer sandwich ELISA transformed from a similar method . In brieIn brief, 20 μl of indicator serum exhibiting high IgE concentrations for Artemisia > 100 kU/l) was incubated with 20 μl of sample serum in the presence of Artemisia pollen extracts  for 1 h at 37 °C, allowing allergen-IgE complex formation. Adding 20 μl of RPMI 1640 medium  instead of sample serum served as control. For complex formation the optimal antibody/allergen ratio was found at concentrations of 1:500 by applying the appropriate indicator serum . It included the symptoms of nasal discharge (rhinorrhea), nasal congestion, itchy nose, sneezing, ocular pruritus, and watery eyes, each with a four-point scale: 0 = no symptoms, 1 = mild symptoms, 2 = moderate symptoms, and 3 = severe symptoms, resulting in a possible total score of 0 to 18. As the symptoms could not be completely alleviated by the AIT treatment, especially when the allergen load was heavy, some rescue medication would be prescribed. The rescue medication score was assessed by the following standards: (1) point for oral or intranasal antihistamines, (2) points for nasal corticosteroids and (3) points for oral corticosteroids. Combined symptom medication scores (SMS) were defined as the sum of RTSS and rescue medication scores .before) and after SCIT (SMSafter) according to aforementioned questionnaire.Subjects were asked to assess the severity of their allergic rhinitis symptoms and the usage of rescue medications at baseline . Analyses were performed by using SPSS 20.0 with P values of less than 0.05 considered as significant, and pictures were created by using Prism software .19 subjects were finally enrolled in the study and their basic information is showed in Table As showed in Table after and serum inhibitory activity for IgE after SCIT, between SMSafter and change of serum inhibitory for IgE were analyzed. However, no significant relationship between serum inhibitory for IgE and clinical improvements was found .The relationship between change of serum inhibitory for IgE during SCIT and clinical improvements, between clinical improvements and serum inhibitory for IgE after SCIT, between SMSWe found that SCIT can effectively relieve allergic symptoms and reduce the use of rescue drugs (P < 0.05), resulting total effective rate as 84.21%. Artemisia-sIgG4 increased significantly after SCIT, and had a moderate correlation with treatment duration , consistent with former studies which reported allergen specific IgG4s could increase in time-dependent manner , 25 duri+ BCRs (B cell receptors) and thus activation of effective T cells, as well as the mediator release of mast cells (MCs) [IgG4s is specific, for it has two different antigen-combining sites, so-called bispecific activity, so it can’t clear allergens efficiently as other immunoglobulins but maybe more effective as an inhibitor of allergen presentation, because it reduces complex size and hence reduces B cell activation . Besidesls (MCs) . So the In the context of allergy, activation of allergic-specific T cells is the key step to induce allergic symptom. Especially, T cell activation mediates late phase reactions by increasing the levels of Th2 cytokines and then recruiting eosinophil and causing tissue damage and remodeling . ReceptoIn addition, CD23 can bind to not only allergen-IgE complexes, but also free IgE and IgE-allergen complexes of different size and composition , which cIt was concluded that Artemisia pollen was the most allergenic pollen in northern part area of Yangtze River in China (with skin test or sIgE blood test) in 2015  and an eThere are also some limitations in this study. First, only one subject was definitely mono-sensitized to Artemisia Table  and all Secondly, the allergen extracts used in detection of sIgG4 and ELIFAB is not the same as the extracts used in SCIT . It was proven by gel electrophoresis that the protein patterns of Artemisia species pollen extracts were similar, with a major band at 24 kDa, like Art v 1 , and it In this paper, we investigated serum inhibitory for IgE as efficacy biomarker for SCIT. However, this study was a preliminary study with a small, poly-sensitized population; therefore, a randomized, double-blind, placebo-controlled study of a large, mono-sensitized population will be needed to evaluate serum inhibitory for IgE as efficacy biomarker for SCIT.Collectively, it was found that serum inhibitory activity for IgE was significantly increased after SCIT and correlated well with serum allergen specific IgG4 levels. However, this change has no clinical relevance in this study, suggesting it’s controversial that serum inhibitory activity for IgE can be used as a biomarker to indicate the efficacy of SCIT, and further research is needed.Additional file 1. It includes the introduction of allergen extract and the best concentration of allergen extract in ELIFAB and the pollen data of Beijing from August 1st to September 29th of each year from 2015 to 2018.