Dendritic Polylysine with Paclitaxel and Triptolide Codelivery for Enhanced Cancer Ferroptosis through the Accumulation of ROS.

Recently, paclitaxel (PTX) was reported to increase intracellular lipid reactive oxygen species (ROS) levels, triggering cancer cell ferroptosis. Based on this, some efforts had been made to improve PTX treatment for non-small-cell lung cancer (NSCLC). Our previous studies demonstrated that triptolide (TPL) could improve the antitumor effect of PTX. Nevertheless, the poor solubility and side effects often limit the application of chemotherapy drugs. In this paper, we constructed a novel nanodrug delivery system (NDDS) chemosynthesis by PEGylated generation 3 (G3) dendritic polylysine coloaded with PTX and TPL (PTX-TPL-PEG-PLL, PTPP), which was endowed with the ability of tumor targeting and favorable solubility. In addition, we demonstrated that TPL could induce ROS generation by regulating the NF-κB signaling pathway to enhance the ferroptosis-induced effect of PTX. Besides, ferroptosis induced by PTPP could improve chemoresistance through inhibiting the level of P-gp, GPX4, and SLC7A11. Furthermore, we determined that ferroptosis may strengthen the immune response by increasing the expression of CD8+ T cells and IFN-γ+ cells while decreasing Treg cells. In general, PTPP may be a potential system for NSCLC treatment.

Oncometabolite 2-hydroxyglutarate regulates anti-tumor immunity.

"Oncometabolite" 2-hydroxyglutarate (2-HG) is an aberrant metabolite found in tumor cells, exerting a pivotal influence on tumor progression. Recent studies have unveiled its impact on the proliferation, activation, and differentiation of anti-tumor T cells. Moreover, 2-HG regulates the function of innate immune components, including macrophages, dendritic cells, natural killer cells, and the complement system. Elevated levels of 2-HG hinder α-KG-dependent dioxygenases (α-KGDDs), contributing to tumorigenesis by disrupting epigenetic regulation, genome integrity, hypoxia-inducible factors (HIF) signaling, and cellular metabolism. The chiral molecular structure of 2-HG produces two enantiomers: D-2-HG and L-2-HG, each with distinct origins and biological functions. Efforts to inhibit D-2-HG and leverage the potential of L-2-HG have demonstrated efficacy in cancer immunotherapy. This review delves into the metabolism, biological functions, and impacts on the tumor immune microenvironment (TIME) of 2-HG, providing a comprehensive exploration of the intricate relationship between 2-HG and antitumor immunity. Additionally, we examine the potential clinical applications of targeted therapy for 2-HG, highlighting recent breakthroughs as well as the existing challenges.

Artificial intelligence-driven design of the assembled major cat allergen Fel d 1 to improve its spatial folding and IgE-reactivity.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSION: 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.

IGF2BP2 Drives Cell Cycle Progression in Triple-Negative Breast Cancer by Recruiting EIF4A1 to Promote the m6A-Modified CDK6 Translation Initiation Process.

IGF2BP2 is a new identified N6-methyladenosine (m6A) reader and associated with poor prognosis in many tumors. However, its role and related mechanism in breast cancer, especially in triple-negative breast cancer (TNBC), remains unclear. In this study, it is found that IGF2BP2 is highly expressed in TNBC due to the lower methylation level in its promoter region. Functional and mechanical studies displayed that IGF2BP2 could promote TNBC proliferation and the G1/S phase transition of the cell cycle via directly regulating CDK6 in an m6A-dependent manner. Surprising, the regulation of protein levels of CDK6 by IGF2BP2 is related to the changes in translation rate during translation initiation, rather than mRNA stability. Moreover, EIF4A1 is found to be recruited by IGF2BP2 to promote the translation output of CDK6, providing new evidence for a regulatory role of IGF2BP2 between m6A methylation and translation. Downregulation of IGF2BP2 in TNBC cell could enhance the sensitivity to abemaciclib, a CDK4/6 inhibitor. To sum up, our study revealed IGF2BP2 could facilitate the translation output of CDK6 via recruiting EIF4A1 and also provided a potential therapeutic target for the diagnosis and treatment of TNBC, as well as a new strategy for broadening the drug indications for CDK4/6 inhibitors.

Identification of Pla a 7 as a novel pollen allergen group in Platanus acerifolia pollen.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The role of matrix stiffness in breast cancer progression: a review.

The significance of matrix stiffness in cancer development has been investigated in recent years. The gradual elastic force the extracellular matrix imparts to cells, known as matrix stiffness, is one of the most important types of mechanical stimulation. Increased matrix stiffness alters the biological activity of cells, which promotes the growth of numerous malignancies, including breast cancer. Comprehensive studies have demonstrated that increasing matrix stiffness activates molecular signaling pathways that are closely linked to breast cancer progression. There are many articles exploring the relationship between mechanism hardness and breast cancer, so we wanted to provide a systematic summary of recent research advances. In this review, we briefly introduce the mechanism of matrix stiffness in breast cancer, elaborate on the effect of extracellular matrix stiffness on breast cancer biological behavior and signaling pathways, and finally, we will talk about breast cancer treatment that focuses on matrix stiffness.

Plant-derived nanovesicles: Further exploration of biomedical function and application potential.

Extracellular vesicles (EVs) are phospholipid bilayer vesicles actively secreted by cells, that contain a variety of functional nucleic acids, proteins, and lipids, and are important mediums of intercellular communication. Based on their natural properties, EVs can not only retain the pharmacological effects of their source cells but also serve as natural delivery carriers. Among them, plant-derived nanovesicles (PNVs) are characterized as natural disease therapeutics with many advantages such as simplicity, safety, eco-friendliness, low cost, and low toxicity due to their abundant resources, large yield, and low risk of immunogenicity in vivo. This review systematically introduces the biogenesis, isolation methods, physical characterization, and components of PNVs, and describes their administration and cellular uptake as therapeutic agents. We highlight the therapeutic potential of PNVs as therapeutic agents and drug delivery carriers, including anti-inflammatory, anticancer, wound healing, regeneration, and antiaging properties as well as their potential use in the treatment of liver disease and COVID-19. Finally, the toxicity and immunogenicity, the current clinical application, and the possible challenges in the future development of PNVs were analyzed. We expect the functions of PNVs to be further explored to promote clinical translation, thereby facilitating the development of a new framework for the treatment of human diseases.

IGF2BP3 mediates the mRNA degradation of NF1 to promote triple-negative breast cancer progression via an m6A-dependent manner.

BACKGROUND: N6-methyladenosine (m6A) is an abundant reversible modification in eukaryotic mRNAs. Emerging evidences indicate that m6A modification plays a vital role in tumourigenesis. As a crucial reader of m6A, IGF2BP3 usually mediates the stabilisation of mRNAs via an m6A-dependent manner. But the underlying mechanism of IGF2BP3 in the tumourigenesis of triple-negative breast cancer (TNBC) is unclear. METHODS: TCGA cohorts were analysed for IGF2BP3 expression and IGF2BP3 promoter methylation levels in different breast cancer subtypes. Colony formation, flow cytometry assays and subcutaneous xenograft were performed to identify the phenotype of IGF2BP3 in TNBC. RNA/RNA immunoprecipitation (RIP)/methylated RNA immunoprecipitation (MeRIP) sequencing and luciferase assays were used to certify the target of IGF2BP3 in TNBC cells. RESULTS: IGF2BP3 was highly expressed in TNBC cell lines and tissues. TET3-mediated IGF2BP3 promoter hypomethylation led to the upregulation of IGF2BP3. Knocking down IGF2BP3 markedly reduced the proliferation of TNBC in vitro and in vivo. Intersection co-assays revealed that IGF2BP3 decreased neurofibromin 1 (NF1) stabilisation via an m6A-dependent manner. NF1 knockdown could rescue the phenotypes of IGF2BP3 knockdown cells partially. CONCLUSION: TET3-mediated IGF2BP3 accelerated the proliferation of TNBC by destabilising NF1 mRNA via an m6A-dependent manner. This suggests that IGF2BP3 could be a potential therapeutic target for TNBC.

Paper-based microfluidics and tailored gold nanoparticles for visual colorimetric detection of multiplex allergens.

The highly efficient and accurate recognition of targeted allergens is an essential element in the diagnosis of allergic diseases and follow-up desensitization treatment in clinic. The current clinical methods widely used to detect sIgE are high cost, time-consuming procedures, and bulky equipment. Herein, a multiplex microfluidic paper-based device (multi-µPAD) was developed that combined with tailored gold nanoparticles for simultaneously visual, colorimetric detection of different allergens in serum. This device could be used as quantitative detection of sIgE with LOD as low as 0.246 KUA/L in colorimetric method. In vitro results also showed that this device possessed good repeatability, high accuracy and incredible stability in different pH (6.0-7.4) and temperature (24-37 °C), as well as long-term storage within 90-day. Finally, this method was successfully utilized for assessing clinical multi-sample screening in 35 allergic patients. After the addition of the samples from allergic patients, the agreement rate of clinical results with commercial enzyme-linked immunosorbent assay (ELISA) kit reached more than 97%, which further indicated that this device had the advantages of efficient, accurate and sensitive to screen various allergens in real clinical serum samples. Therefore, by simply altering antigens and antibodies, this device can also be used for high-throughput detection of other allergens, making it considerable potential for clinical diagnosis of allergic diseases.

Aiming to IgE: Drug development in allergic diseases.

The incidence of allergic disease significantly increases in recent decades, causing it become a major public health problem all over the world. The common allergic diseases such as allergic dermatitis, allergy rhinitis, allergic asthma and food allergy are mediated, at least in part, by immunoglobulin E (IgE), and so IgE acts as a central role in allergic diseases. IgE can interact with its high-affinity receptor (FcεRⅠ) which is primarily expressed on tissue-resident mast cells and circulating basophils, initiating intracellular signal transduction and then causing the activation and degranulation of mast cells and basophils. On the other hand, IgE interaction with its low-affinity receptor (CD23), can regulate various IgE-mediated immune responses including IgE-allergen complex presentation, IgE synthesis, the growth and differentiation of both B and T cells, and the secretion of pro-inflammatory mediators. With the deeper mechanism research for allergic diseases, new therapeutic strategies for interfering IgE are developed and receive a great attention. In this review, we summarize a current profile of therapeutic strategies for interfering IgE in allergic diseases. Besides, we suggest that targeting memory B cells (including long-lived plasma cells and (or) IgE+ memory B cells) may help to completely control allergic diseases, and highlight that the development of drugs synergistically aiming to multiple targets can be a better choice for improving treatment efficacy which results from allergic diseases as the systemic disorders caused by an impaired immune system.

Awakening Allies for Breaking Microenvironment Barriers: NIR-II Guided Orthogonal Activation of Tumor-Infiltrating Mast Cells for Efficient Nano-Drug Delivery.

Mast cells (MCs), powerful immune cells that heavily infiltrate cancer cells, play a crucial role in tumor formation. Activated MCs can release histamine and a family of proteases through degranulation effects, concurrently achieving endothelial junction weakening and stromal degradation of the tumor microenvironment, thereby clearing the obstacles for nano-drug infiltration. To achieve precise activation of tumor-infiltrating MCs, orthogonally excited rare earth nanoparticles (ORENP), with two channels, are introduced for the controllable stimulating drugs release wrapped in "photocut tape". The ORENP can emit near-infrared II (NIR-II) for image tracing for tumor localization in Channel 1 (808/NIR-II) and allows energy upconversion to emit ultraviolet (UV) light for releasing drugs for MCs stimulation in Channel 2 (980/UV). Finally, the combined use of chemical and cellular tools enables clinical nano-drugs to achieve a significant increase in tumor infiltration, thereby enhancing the efficacy of nano-chemotherapy.

Multifunctional Dendritic Au@SPP@DOX Nanoparticles Integrating Chemotherapy and Low-Dose Radiotherapy for Enhanced Anticancer Activity.

Combined chemoradiotherapy can improve antitumor efficiency and reduce the side effects of monotherapy. In this study, we aimed to construct dendritic peptide-based multifunctional nanoparticles (Au@SPP@DOX) for a prolonged circulation time, enhanced cellular uptake, and targeted cancer therapy. Amphiphilic micelle PEG-polylysine-SA (SPP) is composed of polylysine combined with hydrophilic poly(ethylene glycol) (PEG) and hydrophobic stearic acid (SA). Doxorubicin (DOX) is loaded via the hydrophilic-hydrophobic interaction of SPP, and gold nanoparticles (AuNPs) are loaded via the electrostatic interaction with SPP. Au@SPP@DOX showed good biocompatibility and could be successfully accumulated at tumor sites through the enhanced permeability and retention (EPR) effect. Then, lysosomes could be ruptured due to the proton sponge effect. DOX became protonated in response to tumor extracellular acidity and was then released from SPP. Under the action of low-dose radiation, Au@SPP@DOX could promote the production of reactive oxygen species (ROS), increase mitochondrial dysfunction, block cell division, and ultimately promote tumor cell apoptosis to achieve a better antitumor effect. This study highlighted the benefit of chemoradiotherapy and suggested that Au@SPP@DOX might serve as a high-efficiency codelivery system for cancer combination therapy in the future.

Identification and characterization of natural PR-1 protein as major allergen from Humulus japonicus pollen.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 αßα sandwich structure containing 4 α-helices and 6 antiparallel ß-sheets, encompassing 4 conserved CAP domain. CONCLUSION: 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.

Targeting of non-apoptotic cancer cell death mechanisms by quercetin: Implications in cancer therapy.

The ultimate goal of cancer treatment is to kill cancer cells, based on the use of various therapeutic agents, such as chemotherapy, radiotherapy, or targeted therapy drugs. Most drugs exert their therapeutic effects on cancer by targeting apoptosis. However, alterations in apoptosis-related molecules and thus assisting cells to evade death, eventually lead to tumor cell resistance to therapeutic drugs. The increased incidence of non-apoptotic cell death modes such as induced autophagy, mitotic catastrophe, senescence, and necrosis is beneficial to overcoming multidrug resistance mediated by apoptosis resistance in tumor cells. Therefore, investigating the function and mechanism of drug-induced non-apoptotic cell death modes has positive implications for the development of new anti-cancer drugs and therapeutic strategies. Phytochemicals show strong potential as an alternative or complementary medicine for alleviating various types of cancer. Quercetin is a flavonoid compound widely found in the daily diet that demonstrates a significant role in inhibiting numerous human cancers. In addition to direct pro-tumor cell apoptosis, both in vivo and in vitro experiments have shown that quercetin exerts anti-tumor properties by triggering diverse non-apoptotic cell death modes. This review summarized the current status of research on the molecular mechanisms and targets through which quercetin-mediated non-apoptotic mode of cancer cell death, including autophagic cell death, senescence, mitotic catastrophe, ferroptosis, necroptosis, etc.

LnNP@ZIF8 Smart System for In Situ NIR-II Ratiometric Imaging-Based Tumor Drug Resistance Evaluation.

Just-in-time evaluation of drug resistance in situ will greatly facilitate the achievement of precision cancer therapy. The rapid elevation of reactive oxygen species (ROS) is the key to chemotherapy. Hence, suppressed ROS production is an important marker for chemotherapy drug resistance. Herein, a NIR-II emission smart nanoprobe (LnNP@ZIF8, consisting of a lanthanide-doped nanoparticle (LnNP) core and metal-organic framework shell (ZIF8)) is constructed for drug delivery and in vivo NIR-II ratiometric imaging of ROS for tumor drug resistance evaluation. The drug-loaded nanoprobes release therapeutic substances for chemotherapy in the acidic tumor tissue. As the level of ROS increases, the LnNPs shows responsively descending fluorescence intensity at 1550 nm excited by 980 nm (F1550, 980Ex), while the fluorescence of the LnNPs at 1060 nm excited by 808 nm (F1060, 808Ex) is stable. Due to the ratiometric F1550, 980Ex/F1060, 808Ex value exhibiting a linear relationship with ROS concentration, NIR-II imaging results of ROS change based on this ratio can be an important basis for determining tumor drug resistance. As the chemotherapy and resistance evaluation are explored continuously in situ, the ratiometric imaging identifies drug resistance successfully within 24 h, which can greatly improve the timeliness of accurate treatment.

Biomedical Indicators of Patients with Non-Puerperal Mastitis: A Retrospective Study.

BACKGROUND: Non-puerperal mastitis (NPM), a recurrent chronic inflammation of non-lactating breast, often proves tremendous difficulty in treatment, and it may give rise to its complicated symptoms and unclear etiology. Furthermore, the clinical morbidity rate of NPM has been increasing in recent years. METHODS: Overall, 284 patients diagnosed with NPM were consecutively recruited as cases in this study, and patients with benign breast disease (n = 1128) were enrolled as control. The clinical, biomedical, and pathological indicators were analyzed. Univariate and multivariate logistic analysis were used to distinguish risks between NPM and benign breast mass patients. Furthermore, according to the pathological characteristics, the patients of NPM were classified into two subgroups: mammary duct ectasia (MDE) and granulomatous lobular mastitis (GLM). The differences of biomedical indicators between MDE and GLM groups were also analyzed. RESULTS: Compared with benign breast mass group, the level of high-density lipoprotein (HDL-C) significantly decreased, while lipoprotein(a) (Lp(a)) and blood glucose (GLU) both increased in NPM group. According to univariate and multivariate logistic analysis, the onset age and HDL-C were generally decreased, while Lp(a) and GLU were increased in NPM group. The onset age, HDL-C, Lp(a), and GLU were modeled to distinguish NPM and benign breast mass. Significant differences were also observed between MDE and GLM patients in biomedical indicators, such as lipoprotein(a) (Lp(a)), lactate dehydrogenase (LDH), creatine kinase (CK), total cholesterol (TC), and so on. CONCLUSIONS: Our results indicated for the first time that biomarkers were associated with NPM. The biomedical indicators involved in lipid metabolism might be important factors in the development and treatment of NPM. In addition, MDE and GLM are two diseases with different inflammatory states of NPM. These findings would be helpful for a better understanding of NPM and give us some insights to develop new diagnostic and therapeutic strategies.

Purification and characterization of enolase as a novel allergen in Platanus acerifolia pollen.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.