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[Display omitted] • Per a 13 was firstly identified as a novel family of allergen in American cockroach. • The full-length cDNA encoding Per a 13 was firstly isolated. Both nPer a 13 and rPer a 13 were purified and characterized. • Among the patients, 49.3 % were reacted to Per a 13. Patients with allergic rhinitis were more sensitized. Cockroaches are an important source of indoor allergens. Environmental exposure to cockroach allergens is closely associated with the development of immunoglobulin E (IgE)-mediated allergic diseases. However, the allergenic components in the American cockroaches are not fully studied yet. In order to develop novel diagnostic and therapeutic strategies for cockroach allergy, it is necessary to comprehensively investigate this undescribed allergen in the American cockroach. The full-length cDNA of the potential allergen was isolated from the cDNA library of the American cockroach by PCR cloning. Both the recombinant and natural protein molecules were purified and characterized. The allergenicity was further analyzed by enzyme linked immunosorbent assay, immunoblot, and basophil activation test using sera from cockroach allergic patients. A novel allergen belonging to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was firstly identified in the American cockroach and named as Per a 13. The cDNA of this allergen is 1255 base pairs in length and contains an open reading frame of 999 base pairs, encoding 332 amino acids. The purified Per a 13 was fully characterized and assessed to react with IgEs from 49.3 % of cockroach allergic patients, and patients with allergic rhinitis were more sensitized to it. Moreover, the allergenicity was further confirmed by immunoblot and basophil activation test. We firstly identified GAPDH (Per a 13) in the American cockroach, which is a novel type of inhalant allergen derived from animal species. These findings could be useful in developing novel diagnostic and therapeutic strategies for cockroach allergy. [ABSTRACT FROM AUTHOR]

[Display omitted] • A cobalt selenide doped sulfide polyacrylonitrile (CoSe 2 @SPAN) is designed as a cathode for Li-SPAN batteries. • Matched-degree of HOMO level of the nucleophilic reagent S2– and LUMO level of the electrophilic reagent Li+ is significantly improved. • The narrowed energy gap between LUMO and HOMO of CoSe 2 -10@SPAN contributes to the fast reaction kinetics. • Larger Gibbs free energy variation before and after lithiation process promotes the electrochemical reaction. In this work, sulfurized polyacrylonitrile (SPAN) cathodes with atomically dispersed Co and Se active site, namely cobalt selenide doped-sulfide polyacrylonitrile (CoSe 2 - x @SPAN, x = 6, 10, 15) with different CoSe 2 doping concentrations of 6, 10, and 15 wt%, were fabricated by co-heating approach to strengthen the charge conductivity and catalytic activity of SPAN cathode. Atomic scale CoSe 2 were doped into the SPAN skeleton to expediating the redox kinetics of lithium storage process, and the catalytic mechanism was made clear by a viewing angle of frontier molecular orbital theory. The DFT calculation results show that CoSe 2 @SPAN has a more uniform electrostatic potential distribution with a smaller LUMO-HOMO band gap, which is more conducive to electron transport. The Co/Se loaded SPAN polymer constructed by N-Co-S chemical coordination improves the matching degree between the highest occupied molecular orbital (HOMO) of the nucleophile S2− and the lowest unoccupied molecular orbital (LUMO) of the electrophile Li+ during the discharge process, and effectively reduces the bonding orbital σ level of the deposited product Li 2 S, thereby promoting the lithium storage kinetics of CoSe 2 @SPAN cathode material and avoiding the shuttle effect. Meanwhile, the larger Gibbs free energy variation during the lithiation process indicates the enhanced reaction kinetics of the CoSe 2 @SPAN cathode. Ultimately, The Li-SPAN battery with CoSe 2 -10@SPAN cathode delivers high reversible capacity of 1475 mAh g−1 at 0.2 A/g, superior rate capability and long-term capacity retention of 71.1% after 500 cycles at 1.0 A/g. Accordingly, this study offers insights into the utilization of frontier molecular orbital theory (FMO) to improve the redox kinetics and sulfur utilization of Li-SPAN batteries. [ABSTRACT FROM AUTHOR]

• Profilin was one of the major component allergens in P. acerifolia pollen. • The profilin had a significant cross-reactivity with Pop n 2. • Enriching the information on the allergenic components. • Help to develop diagnostic and therapeutic strategies for allergic patients. The Platanus acerifolia (P. acerifolia) pollen is one of the most common causes of allergic respiratory symptoms in China. However, the allergenic components in P. acerifolia are not fully studied yet. The study aimed to determine the molecular and immunochemical characterization of the profilin from P. acerifolia pollen. The coding sequence of profilin was amplified, cloned, and then expressed in Escherichia coli BL21 cells and purified by nickel affinity chromatography. Protein refolding was followed by structural characterization and homology 3D model building. The allergenicity and cross-reactivity were assessed by ELISA, immunoblotting, or basophil activation test (BAT) using the sera of P. acerifolia allergic patients. The cDNA sequence of profilin was cloned with a 396 bp open reading frame coding for 131 amino acids. The molecular weight of the profilin was approximately 14 kDa, and the predicted structure consisted of 3 α-helixes and 7 β-sheets. Physicochemical analysis indicated the profilin was a stable, relatively thermostable, and relatively conserved protein. The allergenicity determined by ELISA, western blot, and BAT suggested 76.9% (30/39) of the P. acerifolia pollen allergic patients displayed specific IgE recognition of the profilin. The profilin shared > 80% sequence identity with Pop n 2, the profilin from Populus nigra , and observed a significant cross-reactivity with Pop n 2 in IgE-inhibition assay. Profilin, as one of the major component allergens in P. acerifolia pollen, was identified and characterized at molecular and immunochemical levels in this study. These findings would contribute to developing diagnostic and therapeutic strategies for P. acerifolia pollen allergic patients. [ABSTRACT FROM AUTHOR]

The Humulus japonicus pollen is one of the most common allergenic pollens in China. However, little is unveiled regarding the allergenic components in Humulus japonicus pollen. Our study aimed to purify and identify the pathogenesis-related 1 (PR-1) protein from Humulus japonicus pollen, and to characterize the molecular and immunochemical properties of this novel allergen. The natural PR-1 protein (named as Hum j PR-1) was purified from Humulus japonicus pollen extracts with a combined strategy of chromatography, and identified by mass spectrometry. The coding sequence of Hum j PR-1 was confirmed by cDNA cloning. The recombinant Hum j PR-1 was expressed and purified from Escherichia coli. The allergenicity was assessed by immunoblot, enzyme-linked immunosorbent assay (ELISA), inhibition ELISA, and basophil activation test using Humulus japonicus allergic patients' whole blood. The physicochemical properties and 3-dimensional structure of it were comprehensively characterized by in silico methods. The allergenicity analysis revealed that 76.6 % (23/30) of the Humulus japonicus pollen allergic patients displayed specific IgE recognition of the natural Hum j PR-1. The cDNA sequence of Hum j PR-1 had a 516-bp open reading frame encoding 171 amino acids. Physicochemical analysis indicated that Hum j PR-1 was a stable and relatively thermostable protein. Hum j PR-1 shared a similar 3-dimensional folding pattern with other homologous allergens, which was a unique αβα sandwich structure containing 4 α-helices and 6 antiparallel β-sheets, encompassing 4 conserved CAP domain. The natural PR-1 was firstly purified and characterized as a major allergenic allergen in Humulus japonicus pollen. These findings would contribute to developing diagnostic and therapeutic strategies for Humulus japonicus pollinosis. • The natural Hum j PR-1 was firstly purified and characterized as a major allergenic allergen in H. japonicus pollen. • Hum j PR-1 showed high sensitization rate of 76.6 % (23/30) in Humulus japonicus pollen-allergic patients. • Hum j PR-1 had a unique αβα sandwich structure with 4 conserved CAP domain. [ABSTRACT FROM AUTHOR]

Long non-coding (lnc) RNAs play crucial functions in human cancer. However, until recently, the involvement of the lncRNA GAS6-AS1 in breast cancer (BCa) malignancy has not been studied exhaustively. The roles and underlying mode of action of GAS6-AS1 action in BCa progression were examined through functional experiments. A decline in GAS6-AS1 level led to a significant decrease in BCa cell proliferation, and the ability for colony formation. Here, GAS6-AS1 competed as endogenous RNA by sequestering microRNA-215-5p (miR-215-5p) causing an enhanced expression of SRY-box transcription factor 9 (SOX9). The effects of silencing GAS6-AS1 on BCa malignant phenotypes could be ameliorated by inhibiting miR-215-5p or restoring SOX9. Thus, GAS6-AS1 acted as a lncRNA that drives tumor in BCa, and enabled progression of BCa through miR-215-5p /SOX9 axis regulation. These outcomes show that the GAS6-AS1/miR-215-5p/SOX9 axis is a potentially effective target for cancer treatment and management. [ABSTRACT FROM AUTHOR]

This study aimed to explore the effects of peroxisome proliferator-activated receptor α (PPAR-α), a known inhibitor of ferroptosis, in Myocardial ischemia/reperfusion injury (MIRI) and its related mechanisms. In vivo and in vitro MIRI models were established. Our results showed that activation of PPAR-α decreased the size of the myocardial infarct, maintained cardiac function, and decreased the serum contents of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and Fe2+ in ischemia/reperfusion (I/R)-treated mice. Additionally, the results of H&E staining, DHE staining, TUNEL staining, and transmission electron microscopy demonstrated that activation of PPAR-α inhibited MIRI-induced heart tissue and mitochondrial damage. It was also found that activation of PPAR-α attenuated MIRI-induced ferroptosis as shown by a reduction in malondialdehyde, total iron, and reactive oxygen species (ROS). In vitro experiments showed that intracellular contents of malondialdehyde, total iron, LDH, reactive oxygen species (ROS), lipid ROS, oxidized glutathione disulphide (GSSG), and Fe2+ were reduced by the activation of PPAR-α in H9c2 cells treated with anoxia/reoxygenation (A/R), while the cell viability and GSH were increased after PPAR-α activation. Additionally, changes in protein levels of the ferroptosis marker further confirmed the beneficial effects of PPAR-α activation on MIRI-induced ferroptosis. Moreover, the results of immunofluorescence and dual-luciferase reporter assay revealed that PPAR-α achieved its activity via binding to the 14-3-3η promoter, promoting its expression level. Moreover, the cardioprotective effects of PPAR-α could be canceled by pAd/14-3-3η-shRNA or Compound C11 (14-3-3η inhibitor). In conclusion, our results indicated that ferroptosis plays a key role in aggravating MIRI, and PPAR-α/14-3-3η pathway-mediated ferroptosis and mitochondrial injury might be an effective therapeutic target against MIRI. [ABSTRACT FROM AUTHOR]

Introduction: Artemisia species are widely spread in north hemisphere. Artemisia sieversiana pollen is one of the common pollen allergens in the north of China. At present, seven allergens were identified and had been listed officially from A. sieversiana pollen, but the remaining allergens are still insufficiently studied, which need to be found. Methods: Pectate lyase was purified from the extracts of A. sieversiana pollen by anion exchange, size exclusion, and HPLC-hydrophobic interaction chromatography. The gene of A. sieversiana pectate lyase (Art si pectate lyase) was cloned and expressed in Escherichia coli. The enzyme activity and circular dichroism (CD) spectrum of natural and recombinant proteins were analyzed. The allergenicity of Art si pectate lyase was characterized by enzyme-linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test. The allergen’s physicochemical properties, three-dimensional structure, sequence profiles with homologous allergens and phylogenetic tree were analyzed by in silico methods. Results: Natural Art si pectate lyase (nArt si pectate lyase) was purified from A. sieversiana pollen extracts by three chromatographic strategies. The cDNA sequence of Art si pectate lyase had a 1191-bp open reading frame encoding 396 amino acids. Both natural and recombinant pectate lyase (rArt si pectate lyase) exhibited similar CD spectrum, and nArt si pectate lyase had higher enzymatic activity. Moreover, the specific immunoglobulin E (IgE) binding rate against nArt si pectate lyase and rArt si pectate lyase was determined as 40% (6/15) in patients’ serum with Artemisia species pollen allergy by ELISA. The nArt si pectate lyase and rArt si pectate lyase could inhibit 76.11% and 47.26% of IgE binding activities to the pollen extracts, respectively. Art si pectate lyase was also confirmed to activate patients’ basophils. Its structure contains a predominant motif of classic parallel helical core, consisting of three parallel β-sheets, and two highly conserved features (vWiDH, RxPxxR) which may contribute to pectate lyase activity. Moreover, Art si pectate lyase shared the highest sequence identity of 73.0% with Art v 6 among currently recognized pectate lyase allergen, both were clustered into the same branch in the phylogenetic tree. Conclusion: In this study, pectate lyase was identified and comprehensively characterized as a novel allergen in A. sieversiana pollen. The findings enriched the allergen information for this pollen and promoted the development of component-resolved diagnosis and molecular therapy of A. sieversiana pollen allergy. [ABSTRACT FROM AUTHOR]

This article discusses the identification and characterization of fructose-bisphosphate aldolase as new pollen allergens. The study found that this allergen was present in Artemisia sieversiana and Artemisia annua pollen and shared similarities with other allergens found in fish and chicken. The researchers purified and characterized the allergen, and it exhibited IgE-binding patterns and allergenicity. The findings suggest that fructose-bisphosphate aldolase could be used for diagnostic and therapeutic purposes in pollen allergies. The study also provides a framework for investigating other unknown pollen allergens. [Extracted from the article]

• We identified and characterized a novel pollen allergen candidate as triosephosphate isomerase, which was different from already known types of pollen allergens and was recognized as Pla a 7 by WHO/IUIS Allergen Nomenclature Sub-committee. • The natural Pla a 7 displayed 41.4% IgE reactivity with the patients' sera, the reactivity was correlated to the IgE concentration against Platanus acerifolia pollen extract. • This finding is essential to enrich information on allergen components and pave the way for molecular diagnosis or treatment strategies for Platanus acerifolia pollen allergy. Platanus acerifolia is recognized as a source of allergenic pollen worldwide. Currently, five Platanus acerifolia pollen allergens belonging to different protein families have been identified, in which profilin and enolase were characterized by our group recently. Besides, we also screened and identified a novel allergen candidate as triosephosphate isomerase, which was different from already known types of pollen allergens. However, the role of this novel allergen group in Platanus acerifolia pollen allergy was unclear. Therefore, we further investigated the allergenicity and clarify its clinical relevance in this study. The natural triosephosphate isomerase from Platanus acerifolia pollen was purified by three steps of chromatography and identified by mass spectrometry. The cDNA sequence of this protein was matched from in-house transcripts based on internal peptide sequences, which was further confirmed by PCR cloning. The recombinant triosephosphate isomerase was expressed and purified from E. coli. Allergenicity analysis of this protein was carried out by enzyme linked immunosorbent assay, immunoblot, and basophil activation test. A novel allergen group belonging to triosephosphate isomerase was firstly identified in Platanus acerifolia pollen and named as Pla a 7. The cDNA of Pla a 7 contained an open reading frame of 762 bp encoding 253 amino acids. The natural Pla a 7 displayed 41.4% IgE reactivity with the patients' sera by ELISA, in which the absorbance value showed correlation to the serum sIgE against Platanus acerifolia pollen extract. Inhibition of IgE-binding to pollen extracts reached 26%-94% in different Pla a 7-positive sera. The recombinant Pla a 7 exhibited weaker IgE-reactivity in ELISA than its natural form, but showed comparable activity in immunoblot. The allergenicity was further confirmed by basophil activation test. Triosephosphate isomerase (Pla a 7) was first recognized as pollen allergen in Platanus acerifolia pollen, which is a completely different type of pollen allergen from those previously reported. This finding is essential to enrich information on allergen components and pave the way for molecular diagnosis or treatment strategies for Platanus acerifolia pollen allergy. [ABSTRACT FROM AUTHOR]

A graphited g -C 3 N 4 assembled with highly-dispersed nickel (HDNi@ g -C 3 N 4) exhibits accelerated reaction kinetics, excellent reversible capacity, and rate performance due to superior metallicity with increased density of states (DOS) at the Fermi energy level, narrowed energy gap between LUMO and HOMO level, and decreased positive Gibbs energy of polysulfide conversion. [Display omitted] • A graphited g -C 3 N 4 assembled with highly-dispersed nickel is designed as a catalyst of Li-S battery. • Exhibiting superior metallicity with increased density of states (DOS) at the Fermi energy level. • Narrowed energy gap contributes much to fast kinetics of negative electrons and positive Li+ ions. • Positive Gibbs energy significantly decreases for the prepared cathode materials. Lithium-sulfur (Li-S) batteries are promising next-generation energy storage devices due to high theoretical energy density and low-cost. Nevertheless, the practical applications are hindered by polysulfide shuttling effect, low electrical conductivity of sulfur, and slower conversion kinetics. Here, the graphited g -C 3 N 4 assembled with highly-dispersed nickel (HDNi@ g -C 3 N 4) is designed as a catalyst to accelerate the reaction kinetics of lithium polysulfide. The oxidized Ni sites of HDNi@ g -C 3 N 4 molecules significantly accommodate the orbital for the electron clouds of polysulfide by forming S n 2–‧‧‧Ni-N active site, thus efficiently improving redox kinetics and mitigating shuttle effects. Based on density functional theory (DFT) calculations, HDNi@ g -C 3 N 4 exhibits a superior metallicity with increased density of states (DOS) at the Fermi energy level. Then, the narrowed energy gap between the lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) level contributes to the enhanced conductivity of catalyst molecular and fast combination between electrons and Li+ ions. Moreover, the positive Gibbs free energy change is significantly decreased for the HDNi@ g -C 3 N 4 cathode. The Li-S battery exhibits a high reversible capacity of 1, 271.6 mAh g−1 at 0.1 C and a high rate capacity of 571.96 mAh g−1 at 2.0 C, a preferable cycling stability with a capacity retention of 53 % even after 500 cycles at a 1.0 C, and an average decay rate of 0.733 % per cycle. [ABSTRACT FROM AUTHOR]

• We purified a new IgE binding protein belonging to enolase family from the extract of P. acerifolia pollen. • We obtained the recombinant form of the protein through expression and purification. • The allergenicity of this novel allergen was characterized by ELISA, Western blot, inhibition ELISA, and basophil activation test. The pollen from Platanus acerifolia (P. acerifolia) is one of the main causes of allergic disorders. To date, only 4 allergens have been identified from this pollen. But previous studies showed that there still exist under-recognized allergens in it. The aim of this study was to comprehensively investigate the newly identified enolase (Pla a 6) as a novel allergen in the P. acerifolia pollen. The natural (n) Pla a 6 was purified by combined chromatographic strategies. According to the identified internal peptides, the cDNA sequence encoding this allergen was matched from the mRNA-sequencing results of P. acerifolia pollen, which was further amplified and cloned. The recombinant (r) Pla a 6 was expressed and purified from E. coli. The allergenicity of this novel allergen was characterized by enzyme linked immunosorbent assay (ELISA), Western blot, inhibition ELISA, and basophil activation test (BAT). A novel allergen from P. acerifolia pollen, named as Pla a 6 was thoroughly studied, which contained an open reading frame of 1338 bp encoding 445 amino acids. The IgE-binding activity of nPla a 6 was initially proved by Western-blot, and a similar IgE-binding pattern to rPla a 6 was also exhibited. Moreover, the positivity for specific IgE against rPla a 6 was tested as 45.95% (17/37) by ELISA, and IgE binding to pollen extract could be inhibited up to 45.77% by 10 µg/ml of rPla a 6. The protein was also confirmed to activate patients' basophils. In this study, a novel allergen belonging to enolase family was comprehensively investigated and characterized through its natural and recombinant forms in P. acerifolia pollen. The study will contribute to the development of novel molecular-based diagnostic and therapeutic approaches for P. acerifolia pollen allergy. [ABSTRACT FROM AUTHOR]

Ischemic cardiomyopathy (ICM) is a serious cardiac disease with a very high mortality rate worldwide, which causes myocardial ischemia and hypoxia as the main damage. Further understanding of the underlying pathological processes of cardiomyocyte injury is key to the development of cardioprotective strategies. Ferroptosis is an iron-dependent form of regulated cell death characterized by the accumulation of lipid hydroperoxides to lethal levels, resulting in oxidative damage to the cell membrane. The current understanding of the role and regulation of ferroptosis in ICM is still limited, especially in the absence of evidence from large-scale transcriptomic data. Through comprehensive bioinformatics analysis of human ICM transcriptome data obtained from the Gene Expression Omnibus database, the present study identified differentially expressed ferroptosis-related genes (DEFRGs) in ICM. Subsequently, their potential biological mechanisms and cross-talk were analyzed, and hub genes were identified by constructing protein-protein interaction networks. Ferroptosis features such as reactive oxygen species generation, changes in ferroptosis marker proteins, iron ion aggregation and lipid oxidation, were identified in the H9c2 anoxic reoxygenation injury model. Finally, the diagnostic ability of Gap junction alpha-1 (GJA1), Solute carrier family 40 member 1 (SLC40A1), Alpha-synuclein (SNCA) were identified through receiver operating characteristic curves and the expression of DEFRGs was verified in an in vitro model. Furthermore, potential drugs (retinoic acid) that could regulate ICM ferroptosis were predicted based on key DEFRGs. The present article presents new insights into the role of ferroptosis in ICM, investigating the regulatory role of ferroptosis in the pathological process of ICM and advocating for ferroptosis as a potential novel therapeutic target for ICM based on evidence from the ICM transcriptome. [ABSTRACT FROM AUTHOR]

Giant ragweed (Ambrosia trifida) pollen is closely associated with respiratory allergy in late summer and autumn, and the prevalence of giant ragweed pollen allergy progressively increases. Compared with short ragweed (Ambrosia artemisiifolia), allergenic components from giant ragweed pollen are poorly investigated. To promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, it becomes necessary to identify and characterize unknown allergens from giant ragweed pollen. In the present study, we identified and characterized a new cysteine-protease (CP) allergen from giant ragweed pollen, named as Amb t CP. The cloned Amb t CP gene encoded 387 amino acids. Recombinant Amb t CP (rAmb t CP) and natural Amb t CP (nAmb t CP) were purified by high-affinity Ni2+ resin and immunoaffinity chromatography respectively. During refolding, purified rAmb t CP could autocatalytically converted to its mature forms displaying a higher enzymatic activity. Moreover, the autocatalytic conversion of proforms to mature forms of nAmb t CP could cause their amount to change in giant ragweed pollen extracts. Then, the allergenicity of Amb t CP was characterized: 23 (33.8%) of 68 Chinese patients with ragweed pollen allergy showed positive IgE binding to nAmb t CP by enzyme-linked immunosorbent assay (ELISA); the result of subsequent ELISA showed that IgE-binding activity of proforms and mature forms of rAmb t CP was different, with positive rate of 39.1% (9/23) and 47.8% (11/23) respectively; Amb t CP showed IgE cross-reactivity with the CP components from short ragweed, Artemisia annua and Artemisia sieversiana pollen. Our findings will help to promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, standardize allergen products and individualize allergen-specific immunotherapy. • A new cysteine protease allergen from Ambrosia trifida pollen, Amb t CP, was identified. • The conversion of pro- to mature Amb t CP depended on a self-activation mechanism. • IgE-binding activity of pro- and mature Amb t CP was different. [ABSTRACT FROM AUTHOR]

• We firstly provided an AI-design strategy to optimize the Fel d 1 with improved spatial conformation between two chains. • The optimized Fel d 1 could fold into its native-like structure with no extra process and its IgE-binding activity was improved. • The strategy lays the foundation for the Fel d 1's industrial production and facilitates its clinical applications for diagnosis. Cat-derived allergens are considered as one of the most common causes of allergic diseases worldwide. Fel d 1 is a major cat allergen and plays an important role in immunoglobulin E (IgE)-reaction diagnosis. However, the two separate chains of Fel d 1 exhibited lower IgE-reactivity than its complete molecule of an assembled form, which makes it difficult to efficiently prepare and limits the application of Fel d 1 in molecular diagnosis of cat allergy. We first applied artificial intelligence (AI) based tool AlphaFold2 to build the 3-dimensional structures of Fel d 1 with different connection modes between two chains, which were evaluated by ERRAT program and were expressed in Escherichia coli. We then calculated the expression ratios of soluble form/inclusion bodies form of optimized Fel d 1. The Circular Dichroism (CD), High Performance Liquid Chromatography-Size Exclusion Chromatography (HPLC-SEC) and reducing/non-reducing SDS-PAGE were performed to characterize the folding status and dimerization of the optimized fusion Fel d 1. The improvement of specific-IgE reactivity to optimized fusion Fel d 1 was investigated by enzyme linked immunosorbent assay (ELISA). Among several linkers, 2 × GGGGS got the highest scores, with an overall quality factor of 100. The error value of the residues around the junction of 2 × GGGGS was lower than others. It exhibited highest proportion of soluble protein than other Fel d 1 constructs with ERRAT (GGGGS, KK as well as direct fusion Fel d 1). The results of CD and HPLC-SEC showed the consistent folding and dimerization of two fused subunits between the optimized fusion Fel d 1 and previously well-defined direct fusion Fel d 1. The overall IgE-binding absorbance of optimized fusion Fel d 1 tested by ELISA was improved compared with that of the direct fusion Fel d 1. We firstly provided an AI-design strategy to optimize the Fel d 1, which could spontaneously fold into its native-like structure without additional refolding process or eukaryotic folding factors. The improved IgE-binding activity and simplified preparation method could greatly facilitate it to be a robust allergen material for molecular diagnosis of cat allergy. [ABSTRACT FROM AUTHOR]

House dust mites are one of the most important allergen sources worldwide and affect approximately 50% of asthmatic patients. Allergen-specific immunotherapy (AIT) is the only disease-modifying treatment for allergic diseases. However, clinical applications of allergen extract-based AIT were greatly restricted due to the potential adverse reactions. In order to improve the efficacy and reduce adverse effects, modified hypoallergens have been proposed for molecular forms of AIT. Therefore, in the present study, we converted the major house dust mite allergen Der f 34 into a B cell epitope-based hypoallergenic vaccine by the immunoinformatics and peptide-carrier fusion approaches. Initially, the physiochemical and structural properties of Der f 34 were analyzed. Accordingly, the linear and conformational B cell epitopes, as well as the helper T lymphocytes epitopes, were computed based on the properties of Der f 34. Three different fragments (residues 12–18, 83–89, and 98–116) of major allergen Der f 34 that containing candidate B cell epitope and that without T cell epitopes were linked at the N terminal and C terminal of the PreS carrier. The three-dimensional structure of the final vaccine was then predicted and the interaction with immune receptors (toll-like receptor-3) was evaluated by ligand-receptor docking. The immunogenic profiles and immune response of the final vaccine were in silico assessed after immunization, which represented the vaccine could induce an effective immune response. In addition, the codon sequences of the vaccine were cloned and expressed in E.coli, the vaccine was purified and exhibited a lower IgE-binding ability. Our results indicated that the Der f 34 hypoallergen could be a potential vaccine candidate for molecular forms of AIT in the house dust mite allergy. [ABSTRACT FROM AUTHOR]