Controlling metal corrosion can directly address the waste of metal and the environmental pollution and resource depletion caused by metal recycling, very significant factors for green and sustainable development. The addition of corrosion inhibitors is a relatively cost-effective means of corrosion prevention. Among these, N-heterocycles have been widely used because heteroatoms contain lone pairs of electrons that can be strongly adsorbed onto metals, protecting them in highly corrosive environments at relatively low concentrations. However, due to the large variety of N-heterocycles, their corrosion inhibition characteristics have seldom been compared; therefore, the selection of appropriate N-heterocycles in the development of anti-corrosion products for specific applications was very difficult. This review systematically analyzed the influence of different substituents on the corrosion inhibition performance of N-heterocycles, including different alkyl chain substituents, electron-donating and electron-withdrawing substituents, and halogen atoms, respectively. The correlation between the molecular structure and corrosion inhibition characteristics of N-heterocycles was comprehensively revealed, and their action mechanism was analyzed deeply. In addition, the toxicity and biodegradability of N-heterocycles was briefly discussed. This study has provided a significant guideline for the development of green, promising corrosion inhibitors for advanced manufacturing and clean energy equipment protection. Metallic materials and alloys can be seen everywhere in production and life. Due to their irreplaceable and excellent mechanical properties, they are used in defense, construction, medicine, energy, transportation and other fields. However, the metals used in manufacturing and life, including iron, copper, aluminum, and others, are commonly confronted with a serious problem: namely, metal corrosion. Corrosion is defined as the chemical and electrochemical reaction of metals with certain specific components in the environment ,2,3,4. MThere are various methods for protection against metal corrosion. These methods generally include changing the metal itself to make an alloy, the cathodic protection method for the sacrificial anode, the applied current negative electrode protection method, the coating method, and the corrosion inhibitor method ,9,10,11.2, -OCH3, -OH, -CHO, -CN, -COO-, -NO2, unsaturated (double bonds and triple bond) bonds and halogen atoms [HSO4], [(CH2)2COOHMIm][HSO4]) for carbon steel in a 0.5 M HCl solution. It was found that ionic liquids can improve the solubility of imidazole rings, and N-heterocyclic parent cations and anions can be adsorbed on metal surfaces as active adsorption sites. The inhibition efficiencies of the above ILs increased with the augment of the alkyl chain length attached to the imidazolium ring, and their favorable performance was ascribed to the formation of IL adsorption films. Moreover, due to the covalent bond formed by charge transfer or sharing and electrostatic attraction, ILs were mainly chemisorbed onto the metallic surface. The longer alkyl chain yielded a stronger interaction of the studied ILs and a larger coverage area on the metal, forming a denser protective film to reduce aggressive attacks.Due to the hydrophobicity, N-heterocyclic corrosion inhibitors with long alkyl chains are somewhat limited in use due to their solubility. The advent of ionic liquids (ILs) has solved the solubility problem and become an excellent alternative in the field of corrosion inhibitors ,45,46,47In order to reveal the action mechanism at the molecular level, the molecular dynamic simulation method was adapted to simulate a large number of N-heterocycles with different alkyl chain lengths. The corrosion inhibition efficiency was evaluated according to the diffusion coefficient of corrosion ions in the corrosion inhibitor film. Three related parameters, that is, the free volume fraction of the film, the interaction energy between the film and the particles and the mobility of the corrosion inhibitor film, were discussed to clarify the diffusion behavior of the corrosive particles in the inhibitor film. Yan  and Yu e(1)When the active group is adsorbed on the metal surface, the longer alkyl chain can increase the coverage and compactness of the corrosion inhibition film. At this time, there are few cavities between the corrosion inhibition films, and the free volume fraction of the corrosion substance is small, slowing down the diffusion coefficient of corrosive ions and increase the corrosion inhibition ability;(2)Due to its electron-donating characteristic, the alkyl chain can change the electron density of the parent body, thereby changing the overall adsorption energy. Therefore, the longer alkyl chain can increase the corrosion inhibitor’s interaction energy with metals, resulting in better corrosion inhibition performance;(3)The longer alkyl chain has greater hydrophobicity, preventing the approach of corrosive ions in water.In conclusion, the alkyl chain length has a great influence on the corrosion inhibition performance of the N-heterocyclic parent. In most cases, the longer the alkyl chain length, the stronger the corrosion inhibition capability of the system. The action mechanism is summarized below:However, due to the type of corrosive medium, temperature, pH, and protective metal, the corrosion inhibition performance of precursors with different chain lengths varies greatly. Even in some specific cases, the alkyl chain length has little effect on the corrosion inhibition properties of the parent. We still need to analyze the situation on a case-by-case basis.+R3, -NO2, trihalomethane , -CN, -SO3H, -CHO, -COR and -COOH. Electron-donating groups are -O-, -NR2, -NHR, -NH2, -OH, -OR, -NHCOR, -OCOR, -R, -CH2COOH and -Ph. In the next section, we discuss the effect of electron-withdrawing and electron-donating substituents on the corrosion inhibition performance of N-heterocyclics. Examples of molecular structures are shown in In most cases, corrosion inhibitors protect the metal by adsorption. In general, N-heterocyclic compounds use the N atom as an active center to form electron sharing or to coordinate lone-pair electrons with empty metal orbitals to achieve a layer of adsorption film for metal protection. The electron-donating and electron-withdrawing abilities of the attached substituent can change the overall electron distribution of the N-heterocycle, thereby affecting the adsorption performance and further affecting the corrosion inhibition ability. It is worth mentioning that whether a group is an electron-withdrawing group or an electron-donating group depends on the sum of its inductive effect, conjugation effect and hyperconjugation effect on the ring. Common electron-withdrawing groups are -Nm represents the meta substituent constant, and σp represents the para substituent constant. Generally, the smaller the σ, the stronger the inhibition effect, which represents a stronger adsorption and higher surface coverage [Ouakki et al.  studied coverage ,21.2COOH (A) > CH2CH2OH (B) > CH2CH2NH2 (C) > H (D)) for carbon steel, which is consistent with the decrease sequential of the Hammett substituent constants as exhibited in −,-Ph) at N1 in 0.3 M NaCl-saturated Ca(OH)2 solution. It was found that the corrosion inhibition effect of the carboxyl substituent (electron-withdrawing group) was better than that of the phenyl substituent (electron donating group).For five-membered N-heterocycles, El-Haddad et al.  found th2OH and -COOC2H5) in HCl for steel. The high inhibition efficiency was explained by the presence of three nitrogen atoms, and electrons of the pyridine and pyrazole ring were the major adsorption centers for their interaction with the metal surface [2OH) of P1 and an electron-withdrawing group (COOC2H5) of P2 by the mesomeric effect to change the density of the molecule. It was found that the substituents did not significantly change the corrosion inhibition mechanism of the main body but did affect the corrosion inhibition efficiency (P1 > P2). Ansari [3 and -OCH3 on the corrosion inhibition performance of compound PC, finding that the electron-donating group reduced the corrosion resistance of the compound instead. It is worth mentioning that the adsorption on the solid surface is not purely physical or chemical, as illustrated in +) adsorbed on the metal surface through electrostatic interaction and formed a protective layer [Regarding pyridine derivatives, Tebbji  studied  surface . The dif. Ansari  evaluateve layer ,69. Fina2+.A six-membered heterocyclic pyrazine containing two N atoms in the para position (AP and ABPT) was investigated by Saha et al. . Based o2) allowed the charge accumulation region to be biased towards Al atoms, while 8-NQ, with the electron-withdrawing group (-NO2), had a bias towards O atoms, which were N and O atoms that provided lone pairs of electrons to form coordination bonds with Al atoms. The above adsorption was performed by the substituent connected to the parent; therefore, the substituent played a vital role in the corrosion inhibition process of the compound. Among the quinoline derivatives, Wang  studied Regarding benzimidazole derivatives, in the study by Aljourani , the the(1)Electron-donating and electron-withdrawing groups affect the adsorption capacity by changing the overall electron density. When the adsorption energy and adsorption area are reduced, the corrosion inhibition ability is also reduced;(2)When the molecular volume of the N-heterocycles is large or very small, the presence of the substituent was negligible for changing the electron distribution of the entire molecule. The addition of electron-donating and electron-withdrawing groups directly changes the number of adsorption sites, at which point the electron-rich heteroatomic centers are directly involved in binding to the metal surface. These electron-rich centers transfer their non-bonded and π electrons to the d orbitals of atoms on the metal surface, forming coordination bonds. At this time, both the electron donating and electron-withdrawing groups increase the corrosion inhibition performance of the compound;(3)In addition, when the substituent changes the hydrophobicity of the entire molecule, the corrosion inhibition efficiency will be affected as well due to the change in solubility of the N-heterocycles. On the one hand, highly hydrophilic compounds may be solvated and cannot adsorb on metal surfaces. On the other hand, highly hydrophobic compounds precipitate due to insoluble polar electrolytes. Both high hydrophilicity and high hydrophobicity will reduce the corrosion inhibition efficiency of the compounds;(4)Besides, the corrosive medium and material of the metal also have a great influence on the corrosion inhibition effect of the compound; therefore, the effect should be discussed according to the specific situation of practical application.The above studies proved that electron-withdrawing and electron-donating groups have a great influence on the corrosion inhibition performance of the N-heterocyclic matrix, although there is no clear correspondence between the electron-donating or electron-withdrawing substituent and the corrosion inhibition ability of N-heterocycle. The law of influence was not fixed and is sometimes even controversial. The action mechanism is summarized below:In general, halogen atoms are a type of electron-withdrawing group; however, they belong to a specific column in the periodic table. For this purpose, we discussed in detail the effects of halogen substituents on the corrosion inhibition behavior of N-heterocycles. Their structural formulas are shown in 2+ on mild steel. As the concentration increased, the coverage of the iron surface increased. The facile dissociation and strong adsorption of 2-I-Imz suggested its potential as an excellent corrosion inhibitor relative to imidazoles and other halogenated imidazoles. During the adsorption process, the parallel conformation of the molecule was more stable due to the large adsorption area with the metal, and the inclined conformation was kinetically unstable in − and Br− corroded by exchanging with the oxygen in the metal-protective oxide film. More pitting damage occurred on the metal surface, with chloride showing the most damage.The adsorption behavior and corrosion inhibition mechanism of C2-halogenated derivatives of imidazole  on an Fe(100) surface were studied by Ismail Abdulazeez et al. . Electro−). Bromine atoms had isolated pairs of electrons that were capable of transmitting them to the d orbital of the Fe and provided the highest inhibition efficiency of BFBT [3) ≈ FA TM(-F). As shown in Chaitra et al.  combined of BFBT . The FNB of BFBT . This de of BFBT  also stuHOMO) and the lowest LUMO energy (ELUMO). The presence of halogens increased the charge on the active atoms of the compound. Sorkhabi et al. [Vidhya  explainei et al.  investigIn addition to substituting single heterocycles, halogens will also replace fused heterocycles. Fernandes et al.  synthesiHOMO, the increase in the ELUMO value, the increase in the energy band gap (ΔE) and the increase in the dipole moment, finally affecting the adsorption ability of the N-heterocycles on the metal surface. The reduced adsorption capacity reduces the coverage on the metal surface, resulting in less protection for the metal. Moreover, the corrosion inhibition performance is related to the stability of the substituents. As the C-halogen bond is easily broken, the dissociated Cl− and Br− may attack the metal surface as corrosive species.In summary, the action mechanism of halogen atoms on the overall corrosion inhibition performance of N-heterocycles is mainly through the electronic effect of halogen substituents. As a rule, the electronegativity of halogen elements refers to the ability of halogen atoms to attract electrons. The larger the electronegativity value of the halogen atom, the stronger the ability of the halogen atom to attract electrons in the compound. In the periodic table of elements, the electronegativity of the halogen elements grows increasingly weak from top to bottom, following F > Cl > Br > I. In general, the corrosion resistance of halogenated compounds is negatively correlated with electronegativity, and their corrosion inhibition properties are generally -I > -Br > -Cl > -F. Taking fluorine substituents as an example: fluorine substituents have a strong electron-withdrawing ability, and the presence of fluorine atoms will consume electron density from the molecular π system, thereby reducing the adsorption activity of adsorption sites. The values of the parameters related to the electronic structure change, such as the decrease in EIt is well known that most N-heterocycles with corrosion inhibitory effects in acid or salt corrosion environments are physical, chemical or mixed adsorption corrosion inhibitors. The adsorption mode of corrosion inhibitors is usually determined by criteria based on the standard adsorption free energy. Values greater than −20 kJ/mol are attributed to physical adsorption, and values less than −40 kJ/mol are attributed to chemisorption, which has been questioned by scientists . HoweverDue to their high corrosion inhibition ability, N-heterocyclic compounds have been used as effective corrosion inhibitors for different metals and alloys. However, with the increasingly strict environmental protection laws and regulations, in order to strengthen ecological environment protection, the use of corrosion inhibitors is becoming more and more strict. Therefore, it is worth paying attention to the toxicity and biodegradability of N-heterocycles.Some nitrogen heterocyclic organic compounds are highly toxic and refractory substances which seriously harm organisms. For example, aromatic N-heterocycles are generally considered to have the “three causes” effects of teratogenicity, carcinogenicity and mutagenesis, which may directly lead to the death of organisms or may exist in organisms to cause genetic mutations of the organisms or flow in the ecological chain, causing wider harm ,86,87,88However, there are few studies on the toxicity and biodegradability of N-heterocycles. Vryzas  and PeddIn order to ensure green and sustainable development, it is important to conduct toxicity and biodegradability studies when developing N-heterocyclic corrosion inhibitors. In the meanwhile, a green and cost-effective synthetic methodology must be considered as well.(1)Under the premise of solubility, the longer the alkyl chain, the stronger the corrosion inhibition ability of N-heterocycles. The alkyl substituent mainly affects the overall anti-corrosion ability by controlling the coverage, density and cavity of the corrosion protection film formed, the adsorption energy and the diffusion coefficient of corrosive ions. Long-chain alkyl substituents with high hydrophobicity increase the distance and difficulty for the corrosive medium to reach the metal;(2)The effect of electron-donating groups and electron-withdrawing groups on the corrosion resistance of N-heterocycles cannot be generalized. In some cases, both electron-donating and electron-withdrawing groups can increase the corrosion inhibition ability of the compound. The electron-donating and electron-withdrawing substituents mainly change the electronic structure of the N-heterocycle and the active site for adsorption, thereby changing the adsorption capacity of the compound and affecting the formation of an adsorption film and the blocking of corrosive media;(3)The influence of a halogen substituent mainly depends on its electronegativity. The overall corrosion inhibition ability of N-heterocycles is negatively correlated with the electronegativity of halogen substituent. The halogen substituents will consume the electron density of N-heterocycles, thereby affecting the overall electron cloud and adsorption capacity, and the dissociated halogen ions may become a new corrosion medium and cause pitting corrosion.In this paper, the inhibitory properties of N-heterocycles with different substituents were reviewed. Corrosion inhibition depends on many factors, including the molecular structure of the inhibitor itself, the number of adsorption sites, the protective metal, the corrosive medium and the ambient temperature. N-heterocycles exhibit different inhibition efficiencies for different metals in different media. All inhibitors prevent corrosion by acting as a barrier layer between the substrate and the environment. Among them, different substituents control the overall corrosion inhibition ability by changing the original hydrophobicity, solubility, electronic structure, molecular volume and active sites of the compound. From our summary and analysis, it can be concluded that:The above analyzed correlations between molecular structure and corrosion inhibition characteristics can act as useful guidelines for the development of new promising N-heterocyclic corrosion inhibitors, especially the study of imidazole, pyridine and triazole compounds. It is believed that an increasing number of N-heterocyclic compounds will be developed and applied because of their advantages in the field of corrosion inhibitors. While in practical research, due to the combined effect of various factors and the specific test conditions, we suggest that the characteristics of each compound be carefully revealed with a combination of experimental tests and computational methods. In situ novel techniques based on the Synchrotron Radiation Facility, such as micro-IR, XANES, etc., and effective theoretical calculation methodologies must be explored to deeply disclose the action mechanism. In theoretical calculation and molecular simulation, there are few studies on the bond length between corrosion inhibitors and metals; therefore, specific calculated values can be added to aid in the effective selection of functional groups. At the same time, molecular structures containing N-heterocyclic can be adjusted according to the corrosion environment to find the most appropriate corrosion inhibitor molecule. In addition, the toxicity and biodegradability of N-containing heterocycles are very important aspects to be considered as well.


Lolium multiflorum grass is the major pollen allergen source in the southern region of Brazil, but most of its allergens remain poorly characterized. The aim of this study was to investigate antibody reactivity to L. multiflorum crude and carboxymethyl-ligand extracts in allergic patients and healthy individuals. Ion exchange carboxymethyl (CM) chromatography (CM-Sepharose) was used to isolate proteins (S2) from L. multiflorum crude extract (S1), which were assessed by SDS-PAGE. S1- and S2-specific IgE and IgG4 levels were measured by ELISA using sera from 55 atopic and 16 non-atopic subjects. Reactive polypeptide bands in S1 and S2 were detected by immunoblotting, and the most prominent bands in S2 were analyzed by mass spectrometry (MS-MS). Similar IgE and IgG4 levels were observed to both S1  and S2  in atopic subjects. S1 and S2 had positive correlations for IgE and IgG4  levels. Homology between S1 and S2 was confirmed by IgE (84%) and IgG4 (83%) inhibition. Immunoblotting revealed that the 29-32 kDa band was recognized by 100% of atopic subjects in both S1 and S2. MS-MS analysis identified similarity profile to groups 1 and 5 grass allergens. This study revealed that carboxymethyl-ligand fraction played an important role for pollen allergy diagnosis by containing clinically relevant allergens and constituted a promising candidate for allergen-specific immunotherapy. Seasonal allergic rhinitis, also known as hay fever, is an upper respiratory disease caused by the inhalation of airborne pollen, affecting allergic people particularly from spring to early summer, with well-defined symptoms, such as nasal itching, nasal congestion, sneezing, runny nose, and conjunctival hyperemia . In the L. multiflorum, Poaceae family, commonly known as Italian ryegrass, was introduced in Brazil by European immigrants, and it has been an important source for sensitizing individuals in the southern region of the country  with high reactivity in patients with allergic rhinitis in subtropical regions, even though it has been responsible for the cross-reactivity with other grasses of the same region  , with no clinical history of pollinosis and negative SPT to L. multiflorum total extract , and subjects signed an informed consent form.A total of 71 subjects were recruited from the southern region of Brazil and distributed in two groups: atopic patients (AT)  with a clinical history of pollinosis and positive skin prick test (SPT) to  extract . The NATDermatophagoides pteronyssinus, Dermatophagoides farinae, and Blomia tropicalis), dog (Canis familiaris), cat dander (Felis domesticus), fungus , and grass pollen  allergen extracts were tested . Histamine hydrochloride 10 mg/mL and 0.9% sodium chloride were used as positive and negative controls, respectively. A wheal size 3 mm greater than the negative control was considered positive. Also, 10 mL of blood was collected from each subject and sera were obtained by centrifugation at 700 g for 10 min at room temperature.Immediate hypersensitivity response was assessed by SPT, as previously described . House dL. multiflorum was identified and registered by the University of Caxias do Sul Herbarium under specimen voucher HUCS 56105 available at https://specieslink.net/search/. Grass pollen was collected in Caxias do Sul, Rio Grande do Sul State, Brazil, during the flowering period of Italian ryegrass (September to November). Pollen content was sieved  to eliminate potential contaminants. After separation, 5 g of grass pollen were mixed in 50 mL of 0.01 M phosphate-buffered saline (PBS), pH 7.2, containing 2 mmol/L phenylmethylsulfonyl fluoride  and submitted to maceration at 4°C and extraction for 18 h at 4°C under orbital stirring. Subsequently, the solution was centrifuged at 15,000 g at 4°C for 30 min and the supernatant was concentrated (S1) and stored at -80°C. The protein content was determined by the Bradford method  (Sigma) for 18 h at 4°C. Carboxymethyl (CM)-Sepharose resin  was packed and equilibrated as recommended by the manufacturer, and extract S1 (2 mg/mL) was loaded onto the column. Unbound proteins were removed by washing with equilibration buffer and bound fractions were eluted with equilibration buffer supplemented with 200 mmol/L NaCl and UV absorbance was measured at 280 nm . The CM-binding fractions with the same electrophoretic profile were pooled (S2) and the protein content of S2 fraction was also determined as previously described.The escribed . BrieflyL. multiflorum were measured by indirect ELISA as previously described (Serum IgE and IgG4 antibodies specific to escribed . High-afToxoplasma gondii (Tg) extract was used as irrelevant antigen. Residual uninhibited antibody reactivity to extract S1 was measured by ELISA as described below and results are reported as the inhibition of S2 fraction in the absence of inhibitors (inhibition %).To evaluate the inhibition of S2 against extract S1, competitive inhibition ELISA was conducted as previously described . High-afTotal extract S1 and fraction S2 were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prepared with 6% polyacrylamide for stacking and 14% for separation, under non-reducing conditions . The gelg for 30 min, the washing solution was discarded, and the steps were repeated 5 times. ACN was added to the gel, vortexed for 5 min, and discarded. Dithiothreitol 10 mmol/L  solution was added to the samples and mixed on a Thermomixer at 56°C at 2200 g for 45 min. Then, the DTT was discarded, followed by the addition of 55 mmol/L iodoacetamide solution . After shaking for 30 min at room temperature in the dark, the IAA solution was discarded. Afterward, the samples were treated with AMBIC (50 mmol/L) and vortexed for 5 min, followed by disposal of the solution. Also, ACN was added to the sample, vortexed for 5 min, and then discarded. The samples were dried in a speed vacuum dryer, added to digestion buffer (100 ng of trypsin in 20 mL of 25 mmol/L AMBIC), and incubated for 16 h at 37°C at 800 g.Liquid chromatography tandem-mass spectrometry (LC-MS/MS) was carried out to identify polypeptide bands. Fraction S2 was subjected to SDS-PAGE as described above, and the bands of 12, 29-32, and 58-69 kDa were excised and stored in 10 mmol/L ammonium bicarbonate . For sample preparation, the solution was discarded and a washing solution  was added; the sample was stirred on a Thermomixer  at 2,200 Peptide extraction was performed with the addition of trifluoroacetic acid  to stop the digestion. The supernatant was collected in a LoBind microtube, mixed with an extraction solution (40% ACN in 0.1% TFA), and stirred for 15 min at room temperature. The process was repeated 4 times. The sample was dried in a speed vacuum dryer and resuspended in 0.1% TFA for desalting. Analyses were performed on LTQ-Orbitrap Velos ETD/Easy nanoLC II (Thermo Fisher Scientific). The peptides were separated on a C18RP column on a 95 min gradient. The instrumental conditions were checked using 100 fmol of a tryptic digest of BSA as standard. The carryover sample was completely removed between runs. The proteins were then filtered by ∼ 10 logp >15, selecting those with >3 unique peptides (UP) or at least 10% equality in the FASTA sequence, according to the UNIPROT database to select highly reliable non-redundant proteins. Alignment sequence analyses were performed using the Basic Local Alignment Search Tool (BLAST), followed by multiple sequence alignment analyses by CLUSTALW.Statistical analysis was performed using the GraphPad Prism software version 6.0 . The frequencies of the antigenic bands recognized by serum antibodies in immunoblotting were subjected to comparative analyses between two proportions by the Z statistic. Kolmogorov-Smirnov normality test was used to determine distribution data model. The Mann-Whitney test was used to compare age, average wheal size, and the ELISA results. Pearson's test was used to determine correlation among data. All results were considered significant at a significance level of P<0.05.2) of 0.996, using cubic curve fit calculated by applying the following equation: Absorbance = 1.575 + 2.499(Conc) - 0.003899[(Conc)2] + 2.025 ×10-6[(Conc)2] + [(Conc)3]. L. multiflorum extract by ion exchange-chromatography. Peak 1 indicates proteins that were not bound to CM-sepharose resin and peak 2 represents resin-binding fractions that were eluted with 200 mmol/L NaCl.Protein content of all samples was determined by the Bradford method using BSA as a standard curve with correlation coefficient  were significantly higher in AT patients  compared to NAT subjects  (P<0.0001) as shown in The levels of serum IgE against L. multiflorum total extract (S1) were significantly higher in AT patients  compared to NAT subjects  (P=0.0019), as shown in The levels of serum IgG4 against IgE and IgG4 cross-reactivity to S1 extract was assessed by inhibition ELISA using S2 fraction. Tg was used as an irrelevant antigen. High homology was observed between extract S1 and fraction S2 for IgE (84%) of pooled sera from the AT group  and for L. multiflorum total extract S1 and fraction S2 components was evaluated in 35 serum samples of AT patients, which were divided into 7 pools (5 serum samples/pool), according to absorbance value obtained in ELISA for specific IgE antibody levels . For negative control, the test was performed in 1 pool with 5 serum samples from NAT subjects. The representative images of immunoblotting for IgE antibody reactivity in AT patients (lanes 2-4) and NAT subjects (lane 5) are shown in The reactivity analysis of L. multiflorum, followed by the 69 kDa band, recognized in 85.7% of the samples. Interestingly, bands of 12, 38, and 58 kDa were recognized by IgE antibodies predominantly in the total extract S1 whereas IgE reactivity to 26 kDa band was predominantly seen in the fraction S2. Other components with variable recognition were the bands ranging from 12 to 145 kDa as shown in The frequency of serum-specific IgE reactivity to the total extract S1 and fraction S2 are reported in L. multiflorum total extract S1 and fraction S2 components in patients AT (lanes 2-4) are illustrated in Also, the presence of specific IgG4 antibodies in AT patients was classified according to the absorbance values obtained in ELISA for specific IgG4 antibodies . For negative control, the test was performed in 1 pool with 5 serum samples from NAT subjects , lane 5.L. multiflorum. Four bands in the extract S1 were exclusively recognized by IgG4 antibodies, namely 18 kDa (100%), 47 kDa (85.7%), 36 kDa (71.4%), and 24 kDa (57.1%). Interestingly, no IgG4 reactivity was seen exclusively to components of fraction S2. Other components with variable recognition were the bands of 43 kDa  and 12 kDa .The frequency-specific IgG4 reactivity to the total extract S1 and fraction S2 components are reported in Oryza sativa  with the sequences obtained from an analysis of the 29-32 kDa band, at least 3 UP sequences were revealed for 2 proteins like those found in other grasses. The first protein found presented 5 UP compatible with Lol p 1, among others β-expansins, and the second protein presented 4 UP sequences compatible with Lol p 5, and 3 other proteins from group 5 , with spa sativa .Lolium multiflorum is one of the main allergen sources that elicit type 2 (T2) inflammatory response in subjects with IgE-mediated hypersensitivity. In the pathophysiological process involved in type I hypersensitivity, allergen proteases from L. multiflorum disrupt the epithelial barrier, thereafter captured by professional antigen presenting cells and presented to naive CD4+ T cells by co-interaction with T cell receptor (TCR) and MHC-II. Those cells will differentiate into Th2 cells by producing interleukin (IL)-5, IL-4, and IL-13 cytokines, which also play an important role in the recruitment of eosinophils and IgE production, which characterize the T2-high endotype   . The IgEg Brazil ,11,27,28Lolium multiflorum, and reacted to pollen allergens from this grass. This was confirmed by SPT, with the average size of wheals larger in the AT group than the NAT group, as expected. Also, IgE- and IgG4-specific antibodies against L. multiflorum were detected using ELISA and immunoblotting techniques only in the AT group. These data are consistent with our previous studies , by mass spectrometry as the major allergen in patients with allergic rhinitis in subtropical regions and responsible for cross-reactivity with other grasses in the same region  since fraction S2 lacks many components that do not have clinical relevance  that may be more appropriate than the total extract (S1) for diagnostic tests such as SPT or for the detection of specific IgE in in vitro assays, and might be a promising candidate for allergen-specific immunotherapy. For all these reasons, we hypothesize that fraction S2 may be a better candidate than the total extract S1 for allergen-specific immunotherapy in seasonal allergic patients sensitized to L. multiflorum. Finally, additional studies should be carried out to assess the effects of fraction S2 compared to the total extract in AIT, since fraction S2 can be derived from a relatively simple method for obtaining a fraction with the natural and genuine major allergens from L. multiflorum pollen.In conclusion, our findings indicated an alternative


The dose and frequency of omalizumab are adjusted based on the patient's body weight and baseline IgE levels, as recommended by its dosing tables. Currently, these dosing recommendations are limited to patients with baseline IgE levels of up to 1500 IU/mL in the European Union and 700 IU/mL in the United States. However, many patients with SAA have IgE levels >1500 IU/mL, highlighting an unmet need. This review presents the current evidence on the treatment benefits of omalizumab in patients with IgE levels >1500 IU/mL. The findings from the reviewed studies which included >3000 patients support the efficacy and effectiveness of omalizumab in reducing exacerbations, and improving asthma control, lung function, and quality of life in patients with severe asthma having IgE levels beyond the current dosing range. Omalizumab was well-tolerated in these patients, with no new safety signals. In addition, high IgE levels (>1500 IU/mL) are also reported in several comorbidities of asthma  and omalizumab has demonstrated efficacy and safety in these indications. These data suggest that omalizumab may be considered for administration in SAA patients, with high IgE levels outside the current dosing tables. A detailed assessment of patients with high IgE levels is needed before deciding on the optimal treatment approach. A management algorithm for SAA patients with IgE >1500 IU/mL is proposed in this review and a suggestion to follow the Delphi consensus is advised.Immunoglobulin E (IgE) plays a critical role in the allergen-initiated inflammatory pathway and thus serves as a viable therapeutic target in allergic or IgE-mediated diseases such as asthma. Omalizumab, an  Severe asthma is one of the most common chronic respiratory disease affecting children and adults worldwide. It is estimated that 3–10% of the global population of 350 million patients with asthma have severe asthma.3,anti-IgE therapy  has a proven efficacy but is limited due to the dosing table of this medication, to a specific range of body weight and baseline blood total IgE levels . This limits the use of omalizumab in SAA patients with total IgE >1500 IU/mL, who are sometimes in great need for such treatment.Asthma is a heterogeneous disease, with allergic asthma being 1 of the most common phenotypes in asthmatic patients, especially in children with severe asthma.The main focus of this review is to discuss the efficacy and safety of omalizumab in patients with asthma with IgE levels >1500 IU/mL. In addition, the article provides an overview on the role of IgE in allergic inflammation, IgE levels in asthma, the rationale for dosing considerations of omalizumab in asthma and a proposed approach for clinical management for these patients. The efficacy of omalizumab in other diseases with high IgE levels is also discussed.naïve T-cells, which subsequently differentiates into allergen-specific Th2 cells. This process consequently leads to B cell-mediated release of IgE, specific to the allergen, which binds to the high-affinity IgE receptor (FcεRI) on mast cells, basophils, and even dendritic cells.,,13IgE plays an important role in allergic diseases such as asthma, atopic dermatitis, and food allergy; it is also known to play a role in non-allergic diseases such as nasal polyposis and chronic spontaneous urticaria.vs 46.65 IU/mL).IgE levels are generally low in plasma (∼0.4 IU/mL),,33Patients with asthma reported increased IgE levels compared to individuals without asthma.,The elevation of IgE in asthma has also been observed in children. In a retrospective chart review conducted in 70 children , 12% of children with asthma had IgE levels >2000 IU/mL.anti-IgE monoclonal antibody in patients aged ≥6 years with uncontrolled moderate-to-severe (United States) and severe (European Union) allergic asthma as an add-on to standard-of-care treatment with high-dose inhaled corticosteroid/long-acting beta-agonist (ICS/LABA).,,,Omalizumab is the only approved ,Initial studies with omalizumab showed that upon administration, it had a very fast effect (within 1 h), reducing free serum IgE by 84%–99% post-single dose.,,Based on these data, a simplified dosing table for omalizumab was developed based on a range of IgE levels and body weights that were thought to be most prevalent in routine clinical practice.,However, the IgE ranges included in the initial and revised omalizumab dosing tables are limited and many studies have reported patients with SAA having IgE levels outside the dosing table limits, including IgE levels >1500 IU/mL.As guidelines on the appropriate dosing of omalizumab for patients with total IgE>1500 are not available per dosing schedule, many patients may not have received omalizumab making them not eligible to receive omalizumab therapy. We performed a literature review to examine efficacy and safety of omalizumab in studies that have administered the drug outside the current dosing table recommendations.A literature search was performed on 14 April 2020 (updated on 10 October 2022) in the PubMed and EMBASE databases (accessed through the Ovid database) using the query string “omalizumab AND asthma AND immunoglobulin E/IgE” to compile a list of studies that reported use of omalizumab in patients with high IgE. The literature search was restricted to studies involving humans and publications in English. A Google search was also performed to identify additional literature . The res,The 9 shortlisted studies comprised clinical trials, observational studies and case reports that included patients with SAA with mean IgE levels of up to 68 628 IU/mL.33The two-year observational eXpeRience registry study involved 925 patients with uncontrolled, persistent asthma who were treated with omalizumab. The study included patients with a range of baseline IgE levels outside the dosing table ; no data on number of patients with IgE >1500 IU/mL included in the study was provided. Overall, omalizumab was associated with reduced exacerbations and OCS use, and improved symptoms and asthma control across all IgE levels.A retrospective, case-control study (26 patients with IgE >700 IU/mL and 26 with IgE 30–700 IU/mL) found that omalizumab was equally safe and effective in reducing corticosteroid use, asthma symptoms and emergency department visits in patients with severe asthma and IgE levels >700 IU/mL  than those with IgE levels 30–700 IU/mL . Patients with IgE levels >700 IU/mL received a mean (± standard deviation [SD]) omalizumab dose of 886 (±220) mg/month.In a fourteen-week randomized clinical trial, patients with asthma were given 450–600 mg omalizumab every 2 weeks, with dose adjusted based on body weight and IgE levels at baseline. Patients with IgE levels between 1200 and 2000 IU/mL were administered 600 mg omalizumab every 2 weeks. Allergen-induced bronchoconstriction and airway inflammation measured by fractioned exhaled nitric oxide (FeNO) were markedly reduced with omalizumab in patients (n = 16) with high IgE levels (700–2000 IU/mL); these patients had a sustained reduction in free IgE levels to <50 ng/mL as early as Week 6.In Australia, the maximum recommended dose of omalizumab in patients with SAA is 750 mg per 4 weeks, as per standard dosing table . However, patients with SAA with IgE >1500 IU/mL and body weight >150 kg (above recommended dosing criteria patients) may also be prescribed with omalizumab at a ceiling dose of 750 mg per 4 weeks. In this analysis of the Australian Xolair® Registry, 179 patients with SAA were analyzed, which included 144 patients within recommended dosing criteria and 55 patients above recommended dosing criteria. Omalizumab was effective in improving asthma symptoms, quality of life, and lung function in patients with SAA above the recommended dosing criteria, with effect comparable to that observed in those within recommended dosing criteria, without the need for dose escalation above 750 mg per 4 weeks.PROSPERO was a multicenter, prospective study in the United States that evaluated the effectiveness and safety of omalizumab in patients with allergic asthma aged ≥12 years over 48 weeks. Nearly 24% of patients had IgE levels above or below those included in the US-based dosing table, with IgE levels up to 68 628.0 IU/mL in adolescents and 8659.2 IU/mL in adults.STELLAIR was a one-year, retrospective, real-life study in French patients with SAA who were treated with omalizumab. IgE levels in the study were in the range of 2–8700 IU/mL at baseline. Omalizumab was associated with treatment effectiveness in the majority of patients: ∼70% of patients rated their response to treatment as "excellent"/"good" on the Global Evaluation of Treatment Effectiveness (GETE) scale, while ∼70% of patients had a ≥40% reduction in annualized exacerbation rate.In a study of 11 children and adolescents being treated at The Children's Hospital of Philadelphia, omalizumab was found to be safe and effective with sustained improvement in asthma control and reduction in hospitalizations in patients with severe asthma and high IgE levels (up to 4320 IU/mL); of note, 7 patients had IgE >1500 IU/mL.In the retrospective REALITY study (131 patients with IgE 30–700 IU/mL and 67 with IgE >700 IU/mL), ∼70% of patients with moderate-to-severe allergic asthma with a mean IgE level of 1046 IU/mL  had a complete response to omalizumab. These patients demonstrated improvements in GETE scores, lung function, and asthma control, and >50% reduction in asthma exacerbations, corticosteroid steroid use, emergency room visits, and hospitalizations.In an analysis of the Severe Asthma Molecular Phenotype cohort from Paris in children (n = 45) with multiple allergic comorbidities and high total IgE levels , omalizumab was found to be highly effective with a complete and rapid response in terms of improvement in asthma control, lung function, and reduction in frequency of severe exacerbations by more than 50%.The effectiveness of omalizumab treatment in these studies involving patients outside the dosing table range  was almost in line with the efficacy/effectiveness observed in patients within the dosing table range. However, it should be taken into consideration that the number of patients with IgE >1500 IU/mL in which effectiveness of omalizumab was assessed is low compared with the overall population of patients. It is noteworthy that none of the studies involving patients with high IgE have reported a lack of response or any specific safety issues related to omalizumab. Overall, these findings were observed in patients with only high IgE levels and in those with both high IgE levels and high body weight, highlighting the efficacy of omalizumab in blocking early asthmatic responses over a broad range of IgE/body weight combinations and downregulating airway inflammation .IgE levels can also reach extremely high levels (>1500 IU/mL) in other diseases that may easily overlap with asthma, which we call co-morbidities or multi-morbidities like atopic dermatitis, allergic bronchopulmonary aspergillosis (ABPA), allergic rhinitis, food allergy and nasal polyposis.Evidence from various clinical trials and case reports has demonstrated the efficacy and safety of omalizumab in patients with ABPA.,anti-IgE pediatric double-blind, placebo-controlled randomized clinical trial (ADAPT), children with atopic dermatitis with median total IgE levels 8373 IU/mL (4556–18506 IU/mL) were treated with omalizumab or placebo for 24 weeks. Of note, 95% of children had baseline total IgE levels >1500 IU/mL. The study reported that children treated with omalizumab showed decrease atopic dermatitis severity and improve QoL despite elevated total IgE levels at baseline.Omalizumab may also be effective in treating patients with atopic dermatitis (off-label use).,Food allergy is frequently associated with allergic asthma and high IgE levels (up to ∼5000 IU/mL),Staphylococcus aureus may lead to increased IgE levels through clonal amplification of IgE positive B cells and sensitization of basophils and mast cells.,S. aureus proteins) may have a potential role in the pathogenesis of asthma, and especially in late-onset asthma, and are associated with disease severity and exacerbations.,Staphylococcus superantigens have also been linked to elevated IgE levels (reaching up to 42 000 IU/mL) and increased severity in patients with atopic dermatitisThe increase in IgE levels in patients with allergic diseases is largely attributed to allergen sensitization. Recent reports suggest that microbial antigens such as enterotoxins from Mycoplasma pneumoniae and Chlamydia pneumoniae are known to be associated with increased asthma exacerbations.Sensitization against mold allergens such as Aspergillus, Alternaria, Cladosporium, Penicillium, and Candida is associated with more severe asthma phenotype requiring multiple hospitalizations.,33,The safety of omalizumab was also evaluated in patients with SAA with IgE >1500 IU/mL and none of the studies have reported safety issues specifically associated with high IgE levels27In the eXpeRience registry (n = 925), patients with asthma with baseline IgE levels 8–7670 IU/mL were treated with omalizumab (as per labelling instructions) for 2 years. Although few serious adverse events (SAEs) were reported, the number of SAEs related to omalizumab was considerably low. Moreover, of nine deaths reported in the registry, none of them were suspected to be related to omalizumab. Overall, no unexpected safety events were reported.In a retrospective study , patients with asthma with mean IgE 221 IU/mL (Group 1) received omalizumab at a mean dose of 426 mg/month (range: 150–900 mg/month), whereas patients with mean IgE 2371 IU/mL (Group 2) received a mean dose of 886 mg/month (range: 400–1200 mg/month) for 6 months or longer duration. No patient reported anaphylaxis or severe injection site reactions. The study results showed that administration of omalizumab outside the therapeutic range showed comparable adverse events (AEs) between Group 1 and Group 2 patients.In a randomized study , asthmatic patients with IgE 30–300 IU/mL (Group 1) and IgE 700–2000 IU/mL (Group 2) were treated with omalizumab (as per dosing table) or placebo for 12–14 weeks. Overall, both omalizumab and placebo were well-tolerated with majority of AEs being mild to moderate in intensity. Nasopharyngitis was the most frequent AE reported. Severe malignant melanoma (Group 2) and breast papilloma (Group 1) were the serious AEs reported, but were not related to study drug. Two patients from the placebo group experienced severe AEs; none of these events were considered study drug related.In a 6-month analysis of Australian Xolair® Registry (n = 179), patients with asthma were grouped based on the dosing table (IgE levels and body weight) into within range and above range and were treated with omalizumab at a maximum dose of 750 mg/month. One patient from each of the groups discontinued omalizumab due to anaphylaxis.In the PROSPERO study, patients with asthma  were treated with omalizumab at a dose of 150 mg every 4 weeks (IgE<30 IU/mL) to 450 mg every 2 weeks (IgE >700 IU/mL) for a median duration of 11.2 months. Of 801 patients who received ≥1 dose of omalizumab, 90 patients experienced 144 S AEs, with the most common SAE being asthma (n = 26/801) and pneumonia (11/801). Although 7 deaths were reported, none of them were suspected to be study drug related. A total of 10 anaphylactic reactions were reported in 4 patients; 3 patients permanently discontinued the drug.In a case series conducted in children aged ≥6 years with IgE levels 892–4320 IU/mL (n = 11), omalizumab treatment (maximum dose: 375 mg every 2 weeks) resulted in no severe reactions. A single patient reported headache and dizziness; however, the patient continued treatment without any further AEs.In the REALITY study (n = 198), asthmatic patients with IgE levels 29–10979 IU/mL who were treated with omalizumab (maximum dose: 600 mg every 2 weeks) for a mean duration of 2.49 years showed no serious AEs.,,,33,Overall, omalizumab has been shown to be well-tolerated in patients with SAA with IgE >1500 IU/mL.28The evidence included in this review confirms the therapeutic efficacy and effectiveness of omalizumab in patients with IgE levels >1500 IU/mL. The pharmacokinetics, pharmacodynamics and safety of omalizumab in patients with IgE levels up to 1500 IU/mL, treated based on the expanded dosing table in 2010, was also found to be comparable to the initially approved dosing table, potentially supporting the tolerability of omalizumab in patients with IgE levels >1500 IU/mL.It should be acknowledged that the studies discussed in this paper were identified following a comprehensive literature search based on search terms of English-language publications; the omission of relevant studies that may not have fit our specific search criteria cannot be discounted. Nevertheless, the evidence from the 9 studies surveyed in this manuscript, which included >3000 patients, support the potential therapeutic benefits of omalizumab in patients with severe asthma with IgE beyond the dosing range. Furthermore, it would be beneficial to conduct a study with different doses of omalizumab in patients with IgE levels >1500 IU/mL to provide more real-world evidence on effectiveness of omalizumab in this population.ex vivo data and further investigation is needed to understand their relevance physiologically. Evidence from a recent in vitro study demonstrated that pre-treatment of plasmocytoid dendritic cells with omalizumab may restore activation of Treg cells, reducing the levels of pro-inflammatory cytokines and supporting extended clinical benefits with this anti-IgE biologic.The efficacy of omalizumab in patients with IgE levels above the limit of the current dosing table, may be supported by its biological activity. Recent studies suggest that in addition to its affinity for free IgE, omalizumab in concentrations >1 μM may also detach IgE from the IgE:FcεR1 complex on basophils, in the absence of Cε2 domain IgE. This may in turn account for the lowered expression of FcεR1 on effector cells in patients treated with omalizumab.Another important consideration for use of omalizumab in patients with asthma with high IgE is the variability in IgE levels over time. Patients with high IgE ineligible based on the dosing table may face increased risk of exacerbations, hospitalizations, and potential side effects from long-term use of OCS. A recent prospective study by Louis and colleaguesThe fluctuation in IgE levels may present a further concern for accurate dosing of patients. The initial dosing levels of patients are determined based on baseline IgE levels; however, as treatment progresses, there may be changes in IgE levels, which may lead to over- or under-dosing. When treatment with omalizumab seems to be less effective in terms of asthma control, monitoring of IgE levels may help tailor treatment and determine whether the patients are receiving the appropriate dosing.Patients with SAA should be assessed at baseline for IgE levels before initiating treatment, and those patients with IgE levels >1500 IU/mL should be evaluated for any comorbidities or any microbial infections that may cause an increase in IgE levels. If there are any comorbidities or infections diagnosed, they should be treated first and then IgE levels should be reassessed. If the patients report persistent high IgE levels, treatment with omalizumab  should be initiated. As the dosing schedule currently gives recommendations on dosing regimen for omalizumab only in patients with IgE ≤1500 IU/mL, Delphi consensus may be required to decide the dose in patients with IgE >1500 IU/mL . In line,Staphylococcus aureus in atopic dermatitis.Omalizumab has shown to be effective in patients with other comorbid allergic conditions such as ABPA, food allergy, nasal polyposis, etc., in whom high IgE levels (IgE >1500 IU/mL) were reported; hence, they should be taken into consideration during the therapeutic management of allergic asthma. Also, as high IgE levels may be mediated by concomitant bacterial and fungal infections,High IgE levels (>1500 IU/mL) are observed in patients with SAA with or without an associated comorbid condition. Omalizumab has been shown in different studies to be an effective and well tolerated option in patients with a broad range of IgE levels reaching up to 67 000 IU/mL, with patients above the current dosing table range receiving comparable therapeutic benefit to patients within the dosing table range.Extending the dosing table IgE range of omalizumab may potentially benefit SAA patients with IgE levels >1500 IU/mL, wherein there is a real and big unmet need. However, patients with IgE levels >1500 IU/mL should be evaluated for comorbidities and infection. If diagnosed, they should be treated first and then IgE levels should be reassessed. A detailed assessment of patients with high IgE levels is needed before deciding on the optimal treatment approach. A management algorithm for SAA patients with IgE >1500 IU/mL is proposed.The maximum doses included in the current dosing tables in the European Union and the United States were the dose ranges validated in the clinical development program. After more than 20 years, many studies worldwide, with many patients having IgE levels >1500 IU/mL, showed effectiveness and a good safety profile for omalizumab with the doses in the current approved tables.Will the highest dosing scheme be enough? Is there a necessity for assessment of new dosing? In the absence of dedicated phase III trials, will a Delphi methodology consensus be enough? Until then, a personalized approach is needed for each patient based on the existing data, the real-world practice and the remaining unmet medical needs. The medical need is out there, it is frequent and real!1, forced expiratory volume in 1 s; FVC, forced vital capacity; GETE, Global Evaluation of Treatment Effectiveness; ICS, inhaled corticosteroid; IgE, immunoglobulin E; LABA, long-acting beta-agonist; OCS, oral corticosteroid; SAA, severe allergic asthma; SAE, serious adverse event; SD, standard deviation; Th2, type 2 helper T cells; US, United States.ABP, allergic bronchoprovocative test; ABP, Aallergic bronchopulmonary aspergillosis; ACT, Asthma Control Test; ACQ, Asthma Control Questionnaire; AD, atopic dermatitis; AE, adverse event; AQLQ, Asthma-related Quality of Life Questionnaire; AR, allergic rhinitis; AUC, area under the curve; EMA, European Medicines Agency; ER, emergency roomEU, European UnionFA, food allergyFDA, Food and Drug AdministrationFeNO, fractional exhaled nitric oxide; FEVhttp://www.ismpp.org/gpp3).The authors would like to acknowledge Dr. Roland Buhl for his valuable insights on this manuscript. Medical writing and editorial support for this manuscript was provided by Archana Jayaraman, Phani Tejasvi Dantu, Preethi B and Rahul Lad of Novartis Healthcare Private Limited, Hyderabad, India, which was funded by Novartis Pharma AG  in accordance with Good Publication Practice (GPP3) guidelines (Not applicable as this is a review article and not an original research.All authors have contributed equally to the design, review, and preparation of the manuscript. All authors have contributed significantly to the work, have read the manuscript, attest to the validity and legitimacy of the data and its interpretation, and agree to its submission.Not applicable, as this is a review article and not an original research.I confirm that all authors have agreed to the submission and consequent publication of the manuscript, post-review by the editorial board.Francesco Menzella has received research grants from AstraZeneca, Novartis Farma, and Sanofi; and lecture fees and advisory board fees from AstraZeneca, Boehringer Ingelheim, Chiesi Farmaceutici, GSK, Mundipharma, Novartis Farma, Angelini, and Sanofi.Jocelyne Just has received consulting fees from Novartis, Sanofi, ALK-Abello, and Astra Zeneca; advisory board and meetings/travel support from Novartis, Sanofi, ALK-Abello, and Astra Zeneca; lecture fees from GSK, Sanofi and Novartis; and grants from Novartis, Astra Zeneca and ALK-Abello.Inessa Schwab Sauerbeck, Claudia Mailaender, Fabiana Saccheri, Celine Thonnelier, Xavier Jaumont and Laurence Mala are employees of Novartis.


KEAP1) and subsequent nuclear factor erythroid 2-related factor 2  activation are strongly associated with ferroptosis resistance in NSCLC. Additionally, AIFM2 gene, which encodes ferroptosis suppressor protein 1 (FSP1), was identified as the gene most significantly correlated with ferroptosis resistance, followed by multiple NRF2 targets. We found that inhibition of NRF2 alone was not sufficient to reduce FSP1 protein levels and promote ferroptosis, whereas FSP1 inhibition effectively sensitized KEAP1-mutant NSCLC cells to ferroptosis. Furthermore, we found that combined inhibition of FSP1 and NRF2 induced ferroptosis more intensely. Our findings imply that FSP1 is a crucial suppressor of ferroptosis whose expression is partially dependent on NRF2 and that synergistically targeting both FSP1 and NRF2 may be a promising strategy for overcoming ferroptosis resistance in cancer.Ferroptosis, a type of cell death induced by lipid peroxidation, has emerged as a novel anti-cancer strategy. Cancer cells frequently acquire resistance to ferroptosis. However, the underlying mechanisms are poorly understood. To address this issue, we conducted a thorough investigation of the genomic and transcriptomic data derived from hundreds of human cancer cell lines and primary tissue samples, with a particular focus on non-small cell lung carcinoma (NSCLC). It was observed that mutations in Kelch-like ECH-associated protein 1 ( Stockwell and coworkers named this process “Ferroptosis” in 2012 , 14KEAP1nt) Fig. . In contnt) Fig. . Lipid pnt) Fig. . Additiont) Fig. . Furthernt) Fig. . HoweverKEAP1 status, we determined the sensitivity of other NSCLC cell lines to RSL3 in the absence or presence of iFSP1. In H2009 and H23 cells, which are KEAP1 wild-type and exhibited low FSP1 expression, no significant synergistic effect of iFSP1 treatment on RSL3-induced ferroptosis was observed  and the farnesoid X receptor (FXR), suggesting the NRF2-independent regulation of FSP1 , 56. NevKEAP1 mutant NSCLC cells, high activity of FSP1, which might be induced by NRF2 or other factors, can rapidly remove lipid peroxyl radicals when initial lipid peroxides accumulate in cells with GPX4 inhibition, thereby preventing the propagation of lipid peroxidation. Consequently, the inhibition of FSP1, in addition to GPX4, allows for the accumulation of lipid peroxides, leading to ferroptosis upon RSL3 treatment. However, the inhibition or depletion of NRF2 was insufficient to effectively reduce the FSP1 protein level, and the remaining FSP1 was adequate to suppress ferroptosis. The supply of ferritin, GSH, and antioxidant proteins by NRF2 may delay continuous lipid peroxidation and amplification. NRF2 inhibition therefore facilitates lipid peroxidation and ferroptosis in cells treated with GPX4 and FSP1 inhibitors. NRF2 and FSP1 are important regulators of ferroptosis, but simultaneous inhibition of NRF2 and FSP1 was not sufficient to induce lipid peroxidation and ferroptosis (Fig. As a result, we suggest that NRF2 and FSP1 suppress ferroptosis via distinct pathways. In sis Fig. , indicatKEAP1 mutations are generally associated with ferroptosis resistance due to the activation of NRF2 and FSP1; however, FSP1 expression may also be regulated independently of KEAP1 and NRF2. As a result, NRF2 activity and FSP1 levels, rather than the KEAP1 genetic status, were clear predictors of ferroptosis resistance. Indeed, FSP1 expression is strongly correlated with ferroptosis sensitivity in a pan-cancer cell line drug sensitivity database. NRF2 inhibition had limited activity toward the reduction of FSP1 and therefore did not have a significant effect on promoting ferroptosis. In conclusion, inhibition of FSP1 with ferroptosis inducers could be a promising therapeutic approach for a wide range of tumors. Furthermore, the concomitant targeting of NRF2 and FSP1 may offer a maximal antitumor effect in KEAP1-mutant tumors.Figure S1Figure S2Figure S3Figure S4Supplementary figure legendUncropped werstern blot


IgE‐epitope profiling can accurately diagnose clinical peanut allergy.We sought to determine whether sequential (linear) epitope‐specific IgE (ses‐IgE) profiling can provide probabilities of tolerating discrete doses of peanut protein in allergic subjects undergoing double‐blind, placebo‐controlled food challenges utilizing PRACTALL dosing.Sixty four ses‐IgE antibodies were quantified in blood samples using a bead‐based epitope assay. A pair of ses‐IgEs that predicts Cumulative Tolerated Dose (CTD) was determined using regression in 75 subjects from the discovery cohort. This epitope‐based predictor was validated on 331 subjects from five independent cohorts (ages 4–25 years). Subjects were grouped based on their predicted values and probabilities of reactions at each CTD threshold were calculated.p < .05); this correlation was 0.51 (p < .05) in validation. Using the ses‐IgE‐based predictor, subjects were assigned into “high,” “moderate,” or “low” dose‐reactivity groups. On average, subjects in the “high” group were four times more likely to tolerate a specific dose, compared with the “low” group. For example, predicted probabilities of tolerating 4, 14, 44, and 144 or 444 mg in the “low” group were 92%, 77%, 53%, 29%, and 10% compared with 98%, 95%, 94%, 88%, and 73% in the “high” group.In discovery, an algorithm using two ses‐IgE antibodies was correlated with CTDs  provide the best prediction of CRD. On average, subjects in the high‐dose reactivity group are 4 times more likely to tolerate a specific dose, compared to the low‐dose group.Abbreviations: CRD, cumulative reactive dose; CTD, cumulative tolerated dose; ses‐IgE, sequential (linear) epitope‐specific immunoglobulin E BBEAbead‐based epitope assayCIconfidence intervalCRDcumulative reactive doseCTDcumulative tolerated doseDBPCFCdouble‐blind, placebo‐controlled food challengeEDeliciting doseIgEimmunoglobulin ELODlimit of detectionMFImedian fluorescence intensityNEGnegative control sampleOFCoral food challengeOIToral immunotherapyPOSpositive control sampleSes‐IgEsequential epitope‐specific IgESPTskin prick test1Accurate diagnosis of food allergy is essential for both minimizing the risk of allergic reactions and eliminating unnecessary dietary restrictions. With a clear‐cut history of allergic reactions following the ingestion of specific foods, the diagnosis is relatively straightforward, while the absence of such history could lead to a challenging multi‐step diagnostic process.A/L is considered a positive result.Ara h 1 and Ara h 2 proteins. Several studies have shown that Ara h 2‐specific IgE is superior to serum IgE to whole peanut in identifying peanut‐allergic subjects.Allergic sensitization can be confirmed by measuring food protein‐specific IgE by either skin prick test (SPT) wheal diameters or serum IgE levels. For peanut, an SPT wheal ≥3 mm or peanut‐specific IgE ≥0.35 kUAra h 2 allergen can identify peanut‐allergic subjects with a sensitivity and specificity >90%.Our group has previously shown that IgE specific to short sequential (linear) epitopes from the 22.1p‐value closest to 1, was selected. To ensure results' validity, a blinding protocol was generated, so that laboratory and clinical data of the Validation cohort could not be integrated until the prediction algorithm was locked.Upon receiving baseline double‐blind, placebo‐controlled food challenge (DBPCFC) data for peanut allergic subjects in BOPI (NCT02149719)During the discovery phase, we were able to obtain additional patient samples from baseline/enrollment DBPCFC of CAFETERIA (NCT03907397), CoFAR6 (NCT01904604),2.2PlateDesigner.Ara h 1 (n = 34), Ara h 2 (n = 16), Ara h 3 (n = 14), with all amino acid sequences published elsewhere.Ara h 2 is the smallest allergen of the three proteins, with 16 epitopes providing a 33% coverage of 49 possible 15‐mer peptides; followed by 17% coverage of the Ara h 1 epitopes (34/205) and 8% for Ara h 3 (14/165).Serum samples from BOPI, OPIA, and PEPITES, and plasma from CAFETERIA and CoFAR6 were randomized across 96‐well plates using Biotinylated peptides were coupled to LumAvidin microspheres (Luminex Corporation) and this master mix was added to 96‐well filter plates. Every plate also included three peptide‐only wells for background quantification (NEG), and three wells with a positive control sample (POS) for the downstream calibration across all plates. The POS sample was composed of a pool of multiple peanut allergic subjects that were not part of this study and serves as a standard control for all BBEA runs; the POS sample is independent of the clinical or any other sample run on the plate. After 3 washes, 100 μl/well of 1:10 diluted plasma or serum samples were added in triplicates and incubated with the peptides for 2 h. After two additional washes, samples were incubated with 50 μl/well of mouse anti‐human phycoerythrin (PE) conjugated IgE  for 30 min. Plates were read on a Luminex‐200 instrument (Luminex Corporation) and IgE for each sample and epitope quantified as a Median Fluorescence Intensity (MFI).s) and epitope‐specific IgE (e) as follows: The MFI was then converted to a “calibrated” value  through the following steps: (1) log2‐transformation, (2) assignment of zero for values below the limit of detection (LOD), and (3) adjusting for inter‐plate variability using the “correction factor.” The median MFI was transformed for each sample (tMFI_POS) on each plate divided by the median of positive controls (tMFI_POS) on plates used in the Discovery phase, specifically BOPI and OPIA studies  and Spearman (rho) correlations were used to measure the linear correlation between the score predicted by each ses‐IgE pair and the actual CRD levels of the patients. The best model was identified, which included IgE to Ara h 2_008 and Ara h 3_100 epitopes. This model was documented and locked; then its performance was evaluated on the Validation samples. Additionally, based on the model's predictor values, patients were split into 3 groups of dose‐reactivity: “low,” “moderate,” or “high” using the following boundaries where a stepwise increase was observed , . Study schematic is outlined in Figure For each pair of ses‐IgEs (2016 combinations), a linear regression was fitted to predict the natural log of cumulative reactive dose (CRD) corresponding to CTDs of the Discovery cohort. Pearson (Ara h 2 (3OB4) and Ara h 3 (3C3V) proteins. However, 6/15 amino acids for Ara h 2_008 and all 15 for Ara h 3_100 were missing from those structures. They were then reconstructed using Swiss‐Model  and visualized using the PyMOL software.Amino acid sequences of Ara h 2_008 and Ara h 3_100 epitopes were mapped to the conformational structures of 2.4p‐values <.00078125 considered significant (Bonferroni correction for 64 tests).CTD values were normalized using natural logarithm (ln). CalMFI values for all 64 ses‐IgEs for patients in the Discovery cohort are presented in a heatmap as a z‐score for each epitope. Using these z‐scores, for each patient we computed a number of IgE‐binding epitopes, that is an epitope is considered “recognized” if z‐score >0. Spearman correlations among ses‐IgEs are presented, with 33.1n = 68), OPIA (n = 56), CAFETERIA (n = 104), CoFAR6 (n = 84), and PEPITES  and OPIA (n = 34) were randomly assigned to the Discovery cohort. The validation cohort included 201 subjects from CAFETERIA, CoFAR6, and the remaining BOPI (n = 27), and OPIA (n = 22) participants. Additionally, all the PEPITES' subjects were used for a final testing of the algorithm.In adherence with the guidelines by the National Academy of Medicine, the development of the CTD algorithm was done in two phases: Discovery and Validation. Seventy‐five subjects from BOPI (3.2p < .001). This means that higher IgE diversity is associated with a lower amount of peanut that a patient can consume without experiencing allergic symptoms. When we binarized a number of IgE‐binding epitopes .Sixty‐four ses‐IgEs were evaluated in 75 subjects in the Discovery cohort Figure , with paIndividual ses‐IgE antibodies had strong pairwise correlation between themselves , as we have observed previously.3.3(s) using only the Discovery cohort:We identified a pair of ses‐IgEs that together provided the best prediction of CRD Table , and devp < .001) in the Discovery cohort, which is higher than correlations of individual antibodies .This algorithm was then documented, locked, and validated on samples and outcome data from 201 subjects which had not been used in any aspect of the discovery process. The performance in this set of previously “unseen” subjects, as expected, was lower, with Spearman correlation of 0.51 , “moderate” (n = 92), and “high” (n = 66).Since CTD values are not truly continuous, generally with threefold increases at each of the escalation doses, and the sample size at each dose tends to be small Figures , a large). On average, subjects in the “high” dose‐reactivity group were 4 times more likely to tolerate a specific dose, compared with the “low” dose group. For example, predicted probabilities of tolerating 4, 14, 44, 144, and 444 mg in the “low” group were 92%, 77%, 53%, 29%, 10% compared with 98%, 95%, 94%, 88%, 73% in the “high” dose‐reactivity group.For each group, we calculated the proportion of subjects that would tolerate different doses of peanut protein Figures  and S3.As an additional validation step of these results, we applied the ses‐IgE‐based algorithm to the PEPITES subjects to obtain their probability distributions. The PEPITES cohort differed from the rest in that all 94 patients had CTDs less than 144 mg, as per eligibility criteria. Ninety‐three percent of the subjects were assigned to either “low” or “moderate” ses‐IgE groups, suggesting that they would have high probability of reacting at low doses, and the distributions of the CTDs in those two groups were similar to that derived from the 237 validation subjects Figure .4DBPCFCs are the “gold standard” for diagnosing food allergy, helping establish reactivity thresholds, and determining if this threshold changes over time or as a result of an intervention.Development of any diagnostic test requires a priori randomization of subjects into discovery and validation sets.We observed that both the levels of individual ses‐IgEs, as well as IgE epitope diversity were inversely associated with the CTD. Allergic effector cells, that is, basophils and mast cells, are saturated with high‐affinity IgE (FceRI) receptors, which upon allergen exposure, IgE molecules on the surface of those cells cross‐link, leading to the release of immune mediators.We set out to develop a predictor using a machine learning approach to evaluate all pairwise ses‐IgE combinations, until the best pair was identified. Two antibodies with the combined strongest association were specific to the Ara h 2_008 and Ara h 3_100 epitopes. Interestingly, the algorithm did not select a pair of IgE epitopes that individually had the highest correlation with the CTD, suggesting an additive effect of these two markers. These IgE epitopes were previously identified as important early predictors of peanut allergy developmentThe CTD predictions were limited by the sample size, since dose increments, in general, follow semi‐log increases and not every dose is equally represented. Additionally, while all the studies followed PRACTALL guidelines, study‐specific dose variations were still present. These factors make CTD predictions less reliable; to address this limitation and make sure that the algorithm can get more precise as more data become available, the outcome of the test was designed to provide a specific probability of reactions at all CTDs. Using the predicted score, patients were assigned to “low,” “moderate,” or “high” dose‐reactor groups, and the probability of tolerating any given dose was fourfold different between the “high” and “low” groups.This risk group assignment is valuable for many purposes, including deciding whether a patient should undergo an OFC to confirm a safe tolerated dose or whether they should maintain stringent allergen avoidance and/or pursue oral immunotherapy (OIT), and to monitor possible allergy resolution over time. For example, a subject with “low” dose‐reactivity group could benefit from OIT.As all samples and outcomes in this study were from the baseline visit of clinical trials, and the subjects were avoiding peanut, this algorithm would need further evaluation to determine its utility for subjects undergoing OIT, where other immune factors could affect the results .In conclusion, this is a first validated algorithm using peanut‐specific epitopes to predict probabilities of reaction to different amounts of peanut in allergic subjects and may provide a useful surrogate for peanut food challenges.The study was conceptualized by P.K., R.G., M.S., and H.S. Sample acquisition and data curation was carried out by C.H., H.B., P.T., and D.K. Data were analyzed and visualized by M.S., P.K., C.H., and H.B; P.T., D.K., S.H.S. were site PIs for BOPI, OPIA, and CAFETERIA studies and provided information and advice regarding participant characterization. M.S. and H.S wrote the original draft. All authors reviewed, edited, and approved the manuscript.The study was funded in part by a grant from National Institute of Allergy and Infectious Diseases , the David H. and Julia Koch Research Program in Food Allergy Therapeutics, and AllerGenis LLC. Food challenges in the BOPI study were funded through a UK Medical Research Council Clinician Scientist award to PJT (reference MR/K010468/1).Maria Suprun has nothing to disclose. Paul Kearney, Clive Hayward, Heather Butler, and Robert Getts are employees of AllerGenis LLC. Dr. Getts has a patent PCT/US15/020715 (WO) pending. Scott H Sicherer reports royalty payments from UpToDate and from Johns Hopkins University Press; grants to his institution from the National Institute of Allergy and Infectious Diseases, and from Food Allergy Research and Education; and personal fees from the American Academy of Allergy, Asthma and Immunology as Deputy Editor of the Journal of Allergy and Clinical Immunology: In Practice, outside of the submitted work. Paul J. Turner reports grants from UK Medical Research Council, NIHR/Imperial Biomedical Research Centre and Jon Moulton Charity Trust; personal fees from UK Food Standards Agency, Aimmune Therapeutics, Allergenis, Aquestive Therapeutics, outside the submitted work. Dianne E Campbell is a part‐time employee of DBV Technologies and reports receiving grant support from National Health and Medical Research Council of Australia and personal fees from Allergenis (Advisory board) and Westmead Fertility Centre (Governance committee). Hugh A Sampson reports grants from Immune Tolerance Network, and NIH‐NIAID, consulting fees from DBV Technologies, N‐Fold, LLC, and Siolta Therapeutics; Mount Sinai has licensed the technology for a bead‐based epitope assay for food‐allergen epitope analysis to AllerGenis LLC, where Dr. Sampson serves as an unpaid Board of Directors member and advisor.Appendix S1Click here for additional data file.


Microtremor recording tests using an accelerometer were carried out in this paper with the aim of characterizing the effects of seasonally frozen soil on the seismic site response, including the two-direction microtremor spectrum, site predominant frequency, and site amplification factor. The study selected eight typical seasonal permafrost sites in China for site microtremor measurements during both summer and winter seasons. Based on the recorded data, the horizontal and vertical components of the microtremor spectrum, HVSR curves, site predominant frequency, and site amplification factor were calculated. The results showed that seasonally frozen soil increased the predominant frequency of the horizontal component of the microtremor spectrum, while the effect on the vertical component was less noticeable. It indicates that the frozen soil layer has a significant impact on the propagation path and energy dissipation of seismic waves in the horizontal direction. Furthermore, the peak values of the horizontal and vertical components of the microtremor spectrum decreased by 30% and 23%, respectively, due to the presence of seasonally frozen soil. The predominant frequency of the site increased by a maximum of 35% and a minimum of 2.8%, while the amplification factor decreased by a maximum of 38% and a minimum of 11%. Additionally, a relationship between the increased site predominant frequency and the cover thickness was proposed. Seasonally frozen soil is a solid freezing layer that forms on the ground during the winter season and is widely distributed in many parts of the world, including the Northeast China, Alaska, and other regions ,2. The cIn recent years, several researchers have investigated the dynamic properties of frozen soil using laboratory and field measures ,5,6. ForWhile laboratory and field measures are the primary methods for investigating the effects of seasonally frozen soil on site seismic response, they are often prohibitively costly and difficult to deploy in urban areas . Given tOne limitation of the one-direction microtremor recording Fourier spectra method is that it presupposes the seismic noise source to be white noise, which is not always true in real-world scenarios. Consequently, this approach can lead to inaccuracies in determining the predominant frequency of the microtremor spectrum, making it challenging to estimate the site amplification factor . On the In seasonally frozen regions, the existence of a frozen layer induces seasonal fluctuations in the mechanical properties of the ground. This variation exerts a substantial influence on the ground motion characteristics of specific sites and, as a result, on the methods employed in seismic design for buildings. The research effort presented in this paper focuses on the effects of seasonally frozen soil on the site seismic response using the microtremor HVSR method. Eight sites in China were selected to represent typical sites with seasonally frozen soils. After that, microtremor measurements were carried out for the eight sites in summer and winter, respectively. The experimental details discussed in this paper may be of interest to researchers in this field. Moreover, the results would expand the application of the HVSR in the urban region and provide references for geotechnical engineering and seismic hazard assessment in areas with frozen soils.sV20 (average shear wave velocities to a depth of 20 m), sV30 (average shear wave velocities to a depth of 30 m), and the thickness of sediments (h) are listed in Eight sites in the northeast and northwest regions of China a were seh (thickness of sediments) of the eight sites varied between 6 and 75 m during the summer. Specifically, two sites in Northwest China had h values of 6 and 11 m, respectively. The sV20 ranged from 165 m/s to 318 m/s, with two sites in Northwest China having sV20 values of 318 m/s and 317 m/s, respectively. The sV30 ranged from 194 m/s to 558 m/s, with two sites in Northwest China having sV30 values of 558 m/s and 520 m/s, respectively.The Microtremor measurements were carried out at eight sites in July and January, respectively. The average temperature in the selected sites during July was 25 °C, the highest monthly average temperature in a year, and the frozen soil had completely melted. Conversely, in January, the temperature reached its lowest point of −25 °C.The TAG-33M accelerometer , which integrates a sensor and a data collector, was used for measuring microtremors in three directions. This accelerometer is ideal for field measurements and other applications, as shown in (a)After selecting the appropriate installation location, use a compass to mark the true north and east directions on the surface; then, drill a D8 mm × 45 mm hole at the installation location using an impact drill a.(b)Insert an M6 × 60 expansion screw into the hole and tighten it with a hammer b.(c)Loosen the nut and place the accelerometer in the center position; then, tighten the nut to secure it onto the accelerometer’s clip.(d)Adjust the orientation of the accelerometer, and then adjust the leveling screws until the bubble is centered c.The installation procedure for recording ground microtremors using the TAG-33M accelerometer is as follows:To ensure reliable microtremor recordings, two instruments  were placed on the same borehole. Each recording lasted 10 min, with a sampling rate of 100 Hz. The unfiltered noise recordings for two instruments  are displayed in n is the number of the time window, n = 1200 s/20.48 s, and i-th time window spectrum for NS, EW, and UD directions.Following the Site Effects Assessment using Ambient Excitations (SESAME) criteria , the sigB) can be estimated from the vibration at the ground surface (S) using Equation (4). h/Vs  in Equation (4) can be derived from Equation (5).To calculate the amplification factor of the eight sites in winter and summer, the estimation method for the site amplification factor proposed by Nakamura  was usedf) ); the T(f) for eight sites is listed in The transfer spectrum can be calculated as the spectral ratio between the ground surface and the basement; then, it is possible to estimate the amplification factor at the predominant frequency of TThe seasonally frozen soil increased the predominant frequency of both the horizontal and vertical components of the microtremor recording Fourier spectrum for the test sites. The average increase in the predominant frequency of the horizontal component was 20.8%, while that of the vertical component was 6%. The difference in the variation in the predominant frequency between the horizontal and vertical components was significant, indicating that the seasonally frozen soil primarily affected the predominant frequency of the horizontal component of the microtremor recording Fourier spectrum, with little impact on the vertical component.(b)The seasonally frozen soil reduced the peak values of both the horizontal and vertical components of the microtremor recording Fourier spectrum for the test sites. The average decrease was 30% for the horizontal component and 23% for the vertical component, with no significant difference between the two. This indicates that the seasonally frozen soil layer had a similar impact on the peak values of the horizontal and vertical components of the microtremor recording Fourier spectrum.(c)The thickness of the sediments and ground types had a significant impact on the influence of the seasonally frozen soil on the predominant frequency of the microtremor recording Fourier spectrum at the test sites. Specifically, at six of the test sites located in Northeast China with cover thicknesses greater than 20 m (class C), the predominant frequency of the horizontal and vertical components of the microtremor recording Fourier spectrum increased, on average, by 0.58 Hz and 0.2 Hz, respectively. However, for the remaining two test sites located in the Gobi sand with cover thicknesses less than 20 m (class B), the predominant frequency of the horizontal and vertical components of the microtremor recording Fourier spectrum increased, on average, by 2.3 Hz and 0.8 Hz.Ibs-von Seth  showed tTherefore, Equations (8) and (9) can be derived, as shown below:h obtained from borehole data, the nonlinear regression fit of Equation (9) is performed, resulting in the derivation of Equations (10) and (11).Using the R2 of Equation (10) was 0.839, which was greater than the critical value of R2 of Equation (11) was 0.774, which is less than 0.834. These results suggest a strong correlation between h is better for the horizontal component of the microtremor recording Fourier spectrum compared to the vertical component.Equations (10) and (11) illustrate the correlation between the increase in the predominant frequency of the horizontal microtremor recording Fourier spectrum and cover thickness, as well as the correlation between the increase in the predominant frequency of the horizontal microtremor recording Fourier spectrum and cover thickness . The AdjHere, H is the horizontal microtremor recording Fourier spectrum, and V is the vertical microtremor recording Fourier spectrum. (a)The predominant frequency of all test sites during the freezing period was found to be higher than that during the nonfreezing period, with the highest increase reaching 35% and the lowest increase at 2.8%. This increase can be attributed to the effect of the seasonally frozen soil on increasing the stiffness of the site.(b)h greater than 40 m, the predominant frequency increment (Δf) was less than 0.1 Hz. Conversely, for the remaining sites with h less than 40 m, the Δf was, on average, 1.5 Hz. Notably, the increase in the predominant frequency was particularly significant for the site with h less than 20 m, suggesting a correlation between h and Δf.For the sites with (c)f and h is examined using Equation (9) and is presented in f and h, with an adjusted R2 value of 0.99. This value is higher than R(8−2)0.001 = 0.925, suggesting that the correlation is statistically significant.The correlation between ΔThe predominant frequency of the test sites (estimated from the peak in the HVSR curve) is listed in f can be estimated using Equation (12).In summary, the results of the study show that the seasonally frozen soil had a significant impact on the predominant frequency of the site and that this impact was inversely proportional to the thickness of the sediments; Δf) curves and amplification factor of eight sites in different seasons are presented in (a)The site amplification factor for all test sites was higher during the nonfreezing period than during the freezing period. The maximum difference was 38% and the minimum difference was 11%. This is likely due to the frozen surface layer dissipating wave motion energy and reducing the amplitude of the site compared to an unfrozen site.(b)The amplification factor of the test sites with a soil surface during the freezing period was significantly lower than during the nonfreezing period, with an average decrease of 33%. However, for the test sites with continuous concrete and asphalt surfaces, the decrease in the amplification factor was relatively small, with an average decrease of 14%. This suggests that the surface layer environment plays a crucial role in the variation in the amplification factor between frozen and unfrozen sites.(c)h and the reduction in the site amplification factor (∆A) was investigated using Equation (8), and the fitting curve is plotted in R2 value of the fitting formula was found to be −0.124, indicating the lack of a significant correlation between h and ∆A.The correlation between The T and (12) were 0.834 and 0.99. It indicates that the correlation between f and h is stronger than the correlation between Hf and h. It may contribute to the fact that the microtremor recording in one direction is susceptible to interference from environmental noise; the HVSR method can more effectively eliminate the impact of such noise compared to recording in only one direction.A subsequent study found that the f values of 0.5 Hz and 0.35 Hz, respectively, and a relative deviation of 43%. Likewise, test sites No. 5 and No. 6 also had comparable soil layer composition and cover thickness, with ∆f values of 0.08 Hz and 0.05 Hz, respectively, and a relative deviation of 60%. The different changes in the site predominant frequency for the similar site characteristics may be attributed to the ground surface environment; the frozen soil layer resulted in a smaller increase in stiffness for the hard surface environment site compared to the soft surface environment site.Test sites No. 3 and No. 4 had similar soil layer composition and cover thickness, but ∆The variation in ∆A appears to be linked to the type of surface environment. For example, in test sites with soil surfaces, the average value of ∆A was 33%. However, in test sites with continuous concrete and asphalt surfaces, the ∆A had an average value of 14%. Additionally, there was no notable correlation found between h and ∆A.The study by Cox B R  demonstrf) was positively correlated with the equivalent shear wave velocity. Therefore, f increases with the presence of frozen soil layers. We assume that these eight sites represent the majority of seasonal frozen soil sites. Therefore, for D-class sites with equivalent shear wave velocities greater than 162 (=180 − 180 × 10%) m/s, C-class sites with velocities greater than 270 (=300 − 300 × 10%) m/s, and B-class sites with velocities greater than 720 (=800 − 800 × 10%) m/s, it is recommended to increase the seismic design level by one grade during the freezing period.id is the thickness of the i-th layer, and iv is the shear wave velocity of the i-th layer.The frozen soil resulted in a 10% increase, on average, in the equivalent shear wave velocity across the eight sites. As indicated by Equation (5), the site predominant frequency ((a)The seasonally frozen soil primarily affected the predominant frequency of the horizontal component of the microtremor recording Fourier spectrum, with little impact on the vertical component.(b)The seasonally frozen soil reduced the peak values of both the horizontal and vertical components of the microtremor recording Fourier spectrum. For the test sites, the average decrease was 30% for the horizontal component and 23% for the vertical component. The seasonally frozen soil layer had a similar impact on the peak values of the horizontal and vertical components of the microtremor recording Fourier spectrum.(c)f had a negative correlation with the thickness of sediments and can be estimated by Equation (12).The predominant frequency of all the test sites during the freezing period was found to be higher than that during the nonfreezing period, with the highest increase reaching 35% and the lowest increase at 2.8%. Δ(d)During the nonfreezing period, the site amplification factor for all the test sites was higher than during the freezing period, with the maximum increase reaching 38% and the minimum increase at 11%. The surface layer environment played a crucial role in the variation in the amplification factor between the frozen and unfrozen sites.A comprehensive investigation of the effects of seasonally frozen soil on site characteristic parameters was carried out and presented in this paper. Eight sites with frozen soil layer thicknesses ranging from 1.5 m to 2.0 m in China were selected to represent typical sites with seasonally frozen soils. After that, microtremor measurements were carried out for the selected sites. For those sites, the conclusions can be summarized as follows: