Mesoscale DNA feature in antibody-coding sequence facilitates somatic hypermutation.

Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities. The CDR hypermutability is mimicable in in vitro deaminase assays and is evolutionarily conserved among species using SHM as a major diversification strategy. We demonstrated that mesoscale sequence alterations tune the in vivo mutability and promote mutations in an otherwise cold region in mice. Our results show a non-coding role of antibody-coding sequence in directing hypermutation, paving the way for the synthetic design of humanized animal models for optimal antibody discovery and explaining the AID mutagenesis pattern in lymphoma.

MACC: a visual interactive knowledgebase of metabolite-associated cell communications.

Metabolite-associated cell communications play critical roles in maintaining the normal biological function of human through coordinating cells, organs and physiological systems. Though substantial information of MACCs has been continuously reported, no relevant database has become available so far. To address this gap, we here developed the first knowledgebase (MACC), to comprehensively describe human metabolite-associated cell communications through curation of experimental literatures. MACC currently contains: (a) 4206 carefully curated metabolite-associated cell communications pairs involving 244 human endogenous metabolites and reported biological effects in vivo and in vitro; (b) 226 comprehensive cell subtypes and 296 disease states, such as cancers, autoimmune diseases, and pathogenic infections; (c) 4508 metabolite-related enzymes and transporters, involving 542 pathways; (d) an interactive tool with user-friendly interface to visualize networks of multiple metabolite-cell interactions. (e) overall expression landscape of metabolite-associated gene sets derived from over 1500 single-cell expression profiles to infer metabolites variations across different cells in the sample. Also, MACC enables cross-links to well-known databases, such as HMDB, DrugBank, TTD and PubMed etc. In complement to ligand-receptor databases, MACC may give new perspectives of alternative communication between cells via metabolite secretion and adsorption, together with the resulting biological functions. MACC is publicly accessible at: http://macc.badd-cao.net/.

SEPPA-mAb: spatial epitope prediction of protein antigens for mAbs.

Identifying the exact epitope positions for a monoclonal antibody (mAb) is of critical importance yet highly challenging to the Ab design of biomedical research. Based on previous versions of SEPPA 3.0, we present SEPPA-mAb for the above purpose with high accuracy and low false positive rate (FPR), suitable for both experimental and modelled structures. In practice, SEPPA-mAb appended a fingerprints-based patch model to SEPPA 3.0, considering the structural and physic-chemical complementarity between a possible epitope patch and the complementarity-determining region of mAb and trained on 860 representative antigen-antibody complexes. On independent testing of 193 antigen-antibody pairs, SEPPA-mAb achieved an accuracy of 0.873 with an FPR of 0.097 in classifying epitope and non-epitope residues under the default threshold, while docking-based methods gave the best AUC of 0.691, and the top epitope prediction tool gave AUC of 0.730 with balanced accuracy of 0.635. A study on 36 independent HIV glycoproteins displayed a high accuracy of 0.918 and a low FPR of 0.058. Further testing illustrated outstanding robustness on new antigens and modelled antibodies. Being the first online tool predicting mAb-specific epitopes, SEPPA-mAb may help to discover new epitopes and design better mAbs for therapeutic and diagnostic purposes. SEPPA-mAb can be accessed at http://www.badd-cao.net/seppa-mab/.

Non-coding RNA in infantile hemangioma.

Infantile hemangioma (IH) is the most common benign vascular tumor of infancy, but its pathogenesis has not been fully discovered. From the cellular perspective, CD133+ stem cells orchestrate the proliferation and development of IH. Regarding molecular mechanisms, hypoxia inducible factor-1α, renin-angiotensin system, and vascular endothelial growth factor are current study hotspots, while non-coding RNAs (ncRNAs) might be essential factors participating in this network. Therefore, this article reviewed published studies concerning the roles of ncRNAs in IH and listed noted miRNAs, lncRNAs, and circRNAs. Other ncRNAs, such as snRNAs, snoRNAs, and tsRNAs, though have not been examined in IH, are mentioned as well to discuss their potential functions. Due to the continuous development of sequencing technologies and computational pipelines for ncRNAs annotation, relevant studies will provide evidence to gradually enhance acknowledgments of ncRNAs' role in IH. The pathogenesis of IH might be revealed and the treatment protocol would be optimized in the future. IMPACT: Non-coding RNAs (ncRNAs) play critical roles in infantile hemangioma. This article thoroughly reviewed all ncRNAs (miRNAs, lncRNAs, and circRNAs) mentioned in previous studies regarding the pathogenesis of infantile hemangioma. Other ncRNAs are promising subjects for further investigation. This review introduced the emerging ncRNAs that need to be explored in IH.

HIT 2.0: an enhanced platform for Herbal Ingredients' Targets.

Literature-described targets of herbal ingredients have been explored to facilitate the mechanistic study of herbs, as well as the new drug discovery. Though several databases provided similar information, the majority of them are limited to literatures before 2010 and need to be updated urgently. HIT 2.0 was here constructed as the latest curated dataset focusing on Herbal Ingredients' Targets covering PubMed literatures 2000-2020. Currently, HIT 2.0 hosts 10 031 compound-target activity pairs with quality indicators between 2208 targets and 1237 ingredients from more than 1250 reputable herbs. The molecular targets cover those genes/proteins being directly/indirectly activated/inhibited, protein binders, and enzymes substrates or products. Also included are those genes regulated under the treatment of individual ingredient. Crosslinks were made to databases of TTD, DrugBank, KEGG, PDB, UniProt, Pfam, NCBI, TCM-ID and others. More importantly, HIT enables automatic Target-mining and My-target curation from daily released PubMed literatures. Thus, users can retrieve and download the latest abstracts containing potential targets for interested compounds, even for those not yet covered in HIT. Further, users can log into 'My-target' system, to curate personal target-profiling on line based on retrieved abstracts. HIT can be accessible at http://hit2.badd-cao.net.

Uptake and cellular responses of Microcystis aeruginosa to PFOS in various environmental conditions.

Although PFOS has been banned as a persistent organic pollutant, it still exists in large quantities within the environment, thus impacting the health of aquatic ecosystems. Previous studies focused solely on high PFOS concentrations, disregarding the connection with environmental factors. To gain a more comprehensive understanding of the PFOS effects on aquatic ecosystems amidst changing environmental conditions, this study investigated the cellular responses of Microcystis aeruginosa to varying PFOS concentrations under heatwave and nutrient stress conditions. The results showed that PFOS concentrations exceeding 5.0 µg/L had obvious effects on multiple physiological responses of M. aeruginosa, resulting in the suppression of algal cell growth and the induction of oxidative damage. However, PFOS concentration at levels below 20.0 µg/L has been found to enhance the growth of algal cells and trigger significant oxidative damage under heatwave conditions. Heatwave conditions could enhance the uptake of PFOS in algal cells, potentially leading to heightened algal growth when PFOS concentration was equal to or less than 5.0 µg/L. Conversely, deficiency or limitation of nitrogen and phosphorus significantly decreased algal abundance and chlorophyll content, inducing severe oxidative stress that could be mitigated by exposure to PFOS. This study holds significance in managing the impact of PFOS on algal growth across diverse environmental conditions.

Endoscopic Repair of Cerebrospinal Fluid Rhinorrhea Due to Trauma by a Bamboo Stick in a Pediatric Patient.

Cerebrospinal fluid rhinorrhea (CSFR) is a condition in which the cerebrospinal fluid flows out of the nasal cavity due to rupture of the arachnoid, dura, and nasal membranes because of bone defects in the skull base. The authors report a rare case of CSFR in a 2-year-old girl who experienced trauma in the nasal cavity by a bamboo stick. She underwent endoscopic repair for the CSFR. During surgery, a bulged vesicle was observed at the left cribriform plate with a small amount of cerebrospinal fluid draining from the surrounding area. Postoperative recovery was good. Endoscopic CSFR repair in pediatric patients is minimally invasive, effective, and safe as demonstrated in this case. Prevention of CSFR in children is important. Parents and caretakers of children need to be more aware, and potentially dangerous objects should not be kept within reach of children.

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Runt-related transcription factor (RUNX1) is a transcription factor closely involved in hematopoiesis. RUNX1 gene mutation plays an essential pathogenic role in the initiation and development of hematological tumors, especially in acute myeloid leukemia. Recent studies have shown that RUNX1 is also involved in the regulation of bone development and the pathological progression of bone-related diseases. RUNX1 promotes the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts and modulates the maturation and extracellular matrix formation of chondrocytes. The expression of RUNX1 in mesenchymal stem cells, chondrocytes, and osteoblasts is of great significance for maintaining normal bone development and the mass and quality of bones. RUNX1 also inhibits the differentiation and bone resorptive activities of osteoclasts, which may be influenced by sexual dimorphism. In addition, RUNX1 deficiency contributes to the pathogenesis of osteoarthritis, delayed fracture healing, and osteoporosis, which was revealed by the RUNX1 conditional knockout modeling in mice. However, the roles of RUNX1 in regulating the hypertrophic differentiation of chondrocytes, the sexual dimorphism of activities of osteoclasts, as well as bone loss in diabetes mellitus, senescence, infection, chronic inflammation, etc, are still not fully understood. This review provides a systematic summary of the research progress concerning RUNX1 in the field of bone biology, offering new ideas for using RUNX1 as a potential target for bone related diseases, especially osteoarthritis, delayed fracture healing, and osteoporosis.

Diphosphine Ligand-Enabled Nickel-Catalyzed Chelate-Assisted Inner-Selective Migratory Hydroarylation of Alkenes.

The precise control of the regioselectivity in the transition metal-catalyzed migratory hydrofunctionalization of alkenes remains a big challenge. With a transient ketimine directing group, the nickel-catalyzed migratory ß-selective hydroarylation and hydroalkenylation of alkenyl ketones has been realized with aryl boronic acids using alkyl halide as the mild hydride source for the first time. The key to this success is the use of a diphosphine ligand, which is capable of the generation of a Ni(II)-H species in the presence of alkyl bromide, and enabling the efficient migratory insertion of alkene into Ni(II)-H species and the sequent rapid chain walking process. The present approach diminishes organosilanes reductant, tolerates a wide array of complex functionalities with excellent regioselective control. Moreover, this catalytic system could also be applied to the migratory hydroarylation of alkenyl azahetereoarenes, thus providing a general approach for the preparation of 1,2-aryl heteroaryl motifs with wide potential applications in pharmaceutical discovery.

A precise molecular subtyping of ulcerative colitis reveals the immune heterogeneity and predicts clinical drug responses.

BACKGROUND AND AIMS: We sought to identify novel molecular subtypes of ulcerative colitis (UC) based on large-scale cohorts and establish a clinically applicable subtyping system for the precision treatment of the disease. METHODS: Eight microarray profiles containing colon samples from 357 patients were utilized. Expression heterogeneity was screened out and stable subtypes were identified among UC patients. Immune infiltration pattern and biological agent response were compared among subtypes to assess the value in guiding treatment. The relationship between PRLR and TNFSF13B genes with the highest predictive value was further validated by functional experiments. RESULTS: Three stable molecular subtypes were successfully identified. Immune cell infiltration analysis defined three subtypes as innate immune activated UC (IIA), whole immune activated UC (WIA), and immune homeostasis like UC (IHL). Notably, the response rate towards biological agents (infliximab/vedolizumab) in WIA patients was the lowest (less than 10%), while the response rate in IHL patients was the highest, ranging from 42 to 60%. Among the featured genes of subtypes, the ratio of PRLR to TNFSF13B could effectively screen for IHL UC subtype suitable for biological agent therapies (Area under curve: 0.961-0.986). Furthermore, we demonstrated that PRLR expressed in epithelial cells could inhibit the expression of TNFSF13B in monocyte-derived macrophages through the CXCL1-NF-κB pathway. CONCLUSIONS: We identified three stable UC subtypes with a heterogeneous immune pattern and different response rates towards biological agents for the first time. We also established a precise molecular subtyping system and classifier to predict clinical drug response and provide individualized treatment strategies for UC patients.

Rank-in: enabling integrative analysis across microarray and RNA-seq for cancer.

Though transcriptomics technologies evolve rapidly in the past decades, integrative analysis of mixed data between microarray and RNA-seq remains challenging due to the inherent variability difference between them. Here, Rank-In was proposed to correct the nonbiological effects across the two technologies, enabling freely blended data for consolidated analysis. Rank-In was rigorously validated via the public cell and tissue samples tested by both technologies. On the two reference samples of the SEQC project, Rank-In not only perfectly classified the 44 profiles but also achieved the best accuracy of 0.9 on predicting TaqMan-validated DEGs. More importantly, on 327 Glioblastoma (GBM) profiles and 248, 523 heterogeneous colon cancer profiles respectively, only Rank-In can successfully discriminate every single cancer profile from normal controls, while the others cannot. Further on different sizes of mixed seq-array GBM profiles, Rank-In can robustly reproduce a median range of DEG overlapping from 0.74 to 0.83 among top genes, whereas the others never exceed 0.72. Being the first effective method enabling mixed data of cross-technology analysis, Rank-In welcomes hybrid of array and seq profiles for integrative study on large/small, paired/unpaired and balanced/imbalanced samples, opening possibility to reduce sampling space of clinical cancer patients. Rank-In can be accessed at http://www.badd-cao.net/rank-in/index.html.

The Evolution Pathway of Ammonia-Oxidizing Archaea Shaped by Major Geological Events.

Primordial nitrification processes have been studied extensively using geochemical approaches, but the biological origination of nitrification remains unclear. Ammonia-oxidizing archaea (AOA) are widely distributed nitrifiers and implement the rate-limiting step in nitrification. They are hypothesized to have been important players in the global nitrogen cycle in Earth's early history. We performed systematic phylogenomic and marker gene analyses to elucidate the diversification timeline of AOA evolution. Our results suggested that the AOA ancestor experienced terrestrial geothermal environments at ∼1,165 Ma (1,928-880 Ma), and gradually evolved into mesophilic soil at ∼652 Ma (767-554 Ma) before diversifying into marine settings at ∼509 Ma (629-412 Ma) and later into shallow and deep oceans, respectively. Corroborated by geochemical evidence and modeling, the timing of key diversification nodes can be linked to the global magmatism and glaciation associated with the assembly and breakup of the supercontinent Rodinia, and the later oxygenation of the deep ocean. Results of this integrated study shed light on the geological forces that may have shaped the evolutionary pathways of the AOA, which played an important role in the ancient global nitrogen cycle.

Yes-associated protein promotes bone healing after tooth extraction in mice.

In this study, a tooth extraction socket model was established in vivo, and Lv-YAP1-GFP, Lv-GFP or saline was injected locally into the extraction socket. Expression of markers of osteogenesis, osteoclastogenesis, adipogenesis, proliferation and apoptosis explore whether YAP can promote bone formation in the process of tooth extraction socket healing. 66 BALB/c mice were divided into 3 groups and underwent left maxillary first molar extraction, Lv-YAP1-GFP, Lv-GFP or saline was injected into the tooth extraction socket. The maxilla was harvested 1, 3, 7, and 10 days after operation for subsequent analysis by Micro-CT and immunohistochemical analysis. Quantitative analysis of the expression of TRAP, ALP, BMP2, RUNX2, Osterix, OCN, RANKL, PPARγ, and PCNA was conducted. The results of immunofluorescence showed that the lentivirus was successfully transfected into the extraction socket. On the middle and last stage of tooth extraction healing, results of Micro-CT showed that the BV/TV, Tb.Th and Tb.N were significantly higher in the experimental group, results of immunohistochemistry showed that the overexpression of YAP increase in the expression of BMP2, ALP, RUNX2, Osterix, OCN, and PCNA. The expression of PPARγ and TUNEL staining results were significantly lower in the experimental group. The expression of TRAP and RANKL showed no significant differences among the 3 groups. We conclude that YAP could promote bone formation in the middle and late stages of tooth extraction socket healing. The overexpression of YAP increased bone formation and cell proliferation, decreased adipogenic differentiation and apoptosis.

Bisphenol A attenuates the therapeutic effect of the selective G protein-coupled estrogen receptor agonist G-1 on allergic rhinitis inflammation in mice.

BACKGROUND: Bisphenol A (BPA) is found in many plastics widely used in everyday life and affects the immune system. Previous studies found that the selective G protein coupled estrogen receptor (GPER) agonist G-1 can reduce the inflammation associated with asthma and allergic rhinitis (AR). BPA also interferes with the protective effect of estradiol against myocardial ischemia-reperfusion injury. OBJECTIVE: We explored whether BPA attenuates the effect of G-1 on inflammation in a mouse AR model. METHODS: The AR model was established by sensitizing and stimulating female BALB/c mice with ovalbumin (OVA) and G-1/BPA. Eosinophils, neutrophils, and lymphocyte subsets (including T and B cells) in nasal mucosa and Th2 and Treg cells in the spleen were detected by flow cytometry. Cytokines and transcription factors characteristic of Th2 and Treg cells in nasal mucosa were detected using cytometric bead arrays and quantitative PCR, respectively. RESULTS: G-1 reduced OVA-induced nasal mucosal inflammation in mice. The proportions of eosinophils, neutrophils, Siglec-F+ neutrophils, lymphocytes, and T cell subsets were reduced by G-1, and this effect was attenuated by BPA. G-1 significantly decreased the Th2 population and levels of IL-4, IL-5, IL-13 and GATA-3; these effects were attenuated by BPA. The enhanced Treg response (as evidenced by an increased Treg population and higher IL-10 and Foxp3 levels) mediated by G-1 tended to be reduced by BPA. DISCUSSION: We found that G-1 reduced OVA-induced nasal mucosal inflammation and significantly decreased the Th2 response, while increasing the Treg response. These effects were attenuated by BPA.

Nonylphenol exacerbates ovalbumin-induced allergic rhinitis via the TSLP-TSLPR/IL-7R pathway and JAK1/2-STAT3 signaling in a mouse model.

Nonylphenol (NP) can be widely used as a plasticizer, surfactant, antioxidant, textile printing, dyeing additive, and pesticide emulsifier. Animal studies have shown that NP aggravates ovalbumin (OVA)-induced allergic rhinitis (AR); however, the exact mechanism underlying its action has not yet been detailed. This study aimed to explore the aggravation of the AR inflammatory response following NP exposure and its possible mechanism. The AR mouse model was constructed using OVA. Under NP exposure, allergic nasal symptoms were observed, eosinophil infiltration was assessed by Sirius red staining, and the levels of IL-4, IL-5, and IL-13 in nasal mucosa samples were detected using cytometric bead array. The mRNA levels of OX40/OX40L and GATA3 in nasal mucosa were detected by qPCR, and the expression levels of the TSLP and JAK1/2-STAT3 signaling pathway components were also identified. Our results suggest that NP exposure exacerbated allergic nasal symptoms and that eosinophils accumulated in nasal mucosa after OVA challenge. The levels of the typical T helper 2 cytokines, as well as the mRNA levels of OX40/OX40L and GATA3, were elevated in the nasal mucosa of OVA-challenged mice exposed to NP. In addition, NP exposure elevated the TSLP, TSLPR, IL-7R, p-JAK1, p-JAK2, and p-STAT3 levels in the nasal mucosa after OVA stimulation. Overall, the present study suggests NP can exacerbate OVA-induced AR inflammatory responses; furthermore, this aggravating effect of NP may be related to the TSLP-TSLPR/IL-7R and JAK1/2-STAT3 signaling pathways.

Dermoid Cyst of Nasal Tip and Nasal Septum.

ABSTRACT: Dermoid cyst is a congenital and benign disease with most occur on the head and neck. It is rarely that occur on the nasal tip and nasal septum at same time and rarely repair of using nasal septum mucosa. The authors treated a child with dermoid cyst in the nasal tip and septum. Only the dermoid cyst at the tip of the nose caused the change of appearance. Dermoid cyst of nasal septum did not cause any clinical symptoms. She underwent excision of the dermoid cyst at the tip of the nose and endoscopic surgery for the dermoid cyst in the nasal septum and used the nasal septum mucosa for repair at the same time. After 6 months of recovery, the appearance of the nasal tip recovered well without obvious scar, the nasal septum area recovered well, and the local stoma was unobstructed without recurrence. The authors found that this kind of nasal septal cyst with no clinical symptoms can achieve good therapeutic effect through endoscopic surgery and repair of using nasal septum mucosa, with less damage, rapid recovery, and good prognosis.

Endoscopic Resection of a Rare Chondromyxoid Fibroma From the Nasal Septum.

ABSTRACT: Chondromyxoid fibroma is a rare benign tumor mostly found in the metaphysis of long bones. In rare cases, it develops in unusual locations. We report a case of chondromyxoid fibroma from the nasal septum. Endoscopic surgery was performed with the patient under general anesthesia. A plasma knife was used to ablate the tumor. No recurrence was noted at the 4-year follow-up. The advantages of endoscopic surgery include direct observation, improved visibility and magnification, reduced intraoperative trauma, and fewer postoperative complications. The advantages of the plasma knife include its ability to separate and ablate the tumor simultaneously while effectively reducing bleeding and maintaining the visibility of the surgical field.

Targeting a novel hsa_circ_0000520/miR-556-5p/NLRP3 pathway-mediated cell pyroptosis and inflammation attenuates ovalbumin (OVA)-induced allergic rhinitis (AR) in mice models.

OBJECTIVES: The circRNAs-miRNAs-mRNAs competing endogenous RNA (ceRNA) networks involve in regulating the development of various inflammation-associated diseases, including allergic rhinitis (AR), and the present study aimed to identify novel AR-associated ceRNA networks. METHODS: The mRNA and protein levels of the associated genes were, respectively, examined by real-time qPCR and western blot analysis. The targeting sites in miR-556-5p and NLRP3 were validated by performing dual-luciferase reporter gene system assay. ELISA was used to measure inflammatory cytokines secretion, and CCK-8 assay was conducted to determine cell proliferation. RESULTS: Here, we first identified a hsa_circ_0000520/miR-556-5p/NLRP3 signaling cascade triggered epithelium pyroptosis and inflammation to regulate the development of AR in cellular and mice models. Specifically, the pyroptosis-associated biomarkers (NLRP3, ASC, IL-1ß and IL-18) and pro-inflammatory cytokines (OVA-specific IgE, TNF-α, IL-4 and IL-5) were upregulated in the nasal subjects collected from AR patients and ovalbumin (OVA)-induced AR mice models, compared to their normal counterparts. Next, using the ceRNA networks analysis software, we screened out a hsa_circ_0000520/miR-556-5p axis that potentially regulated NLRP3 in the human nasal epithelial cell line. Mechanistically, miR-556-5p targeted both hsa_circ_0000520 and 3' untranslated region (3'UTR) of NLRP3, and knock-down of hsa_circ_0000520 inactivated NLRP3-mediated epithelium pyroptosis through miR-556-5p in a ceRNA-dependent manner. Furthermore, we proved that both hsa_circ_0000520 ablation and miR-556-5p overexpression suppressed NLRP3-mediated cell pyroptosis to attenuate AR in mice models. CONCLUSIONS: Taken together, we evidenced that targeting the hsa_circ_0000520/miR-556-5p/NLRP3 signaling pathway was a novel AQ1strategy to ameliorate AR progression; however, future clinical data are still required to validate our preliminary results.

Retracted: Protective Effect of Circular RNA (CircRNA) Ddx17 in Ovalbumin (OVA)-Induced Allergic Rhinitis (AR) Mice.

On the authors' request due to a need for verification and data supplementation. Reference: Jiao Liu, Zhiwei Cao: Protective Effect of Circular RNA (CircRNA) Ddx17 in Ovalbumin (OVA)-Induced Allergic Rhinitis (AR) Mice.  Med Sci Monit 2020; 26:e919083. 10.12659/MSM.919083.

SEPPA 3.0-enhanced spatial epitope prediction enabling glycoprotein antigens.

B-cell epitope information is critical to immune therapy and vaccine design. Protein epitopes can be significantly affected by glycosylation, while no methods have considered this till now. Based on previous versions of Spatial Epitope Prediction of Protein Antigens (SEPPA), we here present an enhanced tool SEPPA 3.0, enabling glycoprotein antigens. Parameters were updated based on the latest and largest dataset. Then, additional micro-environmental features of glycosylation triangles and glycosylation-related amino acid indexes were added as important classifiers, coupled with final calibration based on neighboring antigenicity. Logistic regression model was retained as SEPPA 2.0. The AUC value of 0.794 was obtained through 10-fold cross-validation on internal validation. Independent testing on general protein antigens resulted in AUC of 0.740 with BA (balanced accuracy) of 0.657 as baseline of SEPPA 3.0. Most importantly, when tested on independent glycoprotein antigens only, SEPPA 3.0 gave an AUC of 0.749 and BA of 0.665, leading the top performance among peers. As the first server enabling accurate epitope prediction for glycoproteins, SEPPA 3.0 shows significant advantages over popular peers on both general protein and glycoprotein antigens. It can be accessed at http://bidd2.nus.edu.sg/SEPPA3/ or at http://www.badd-cao.net/seppa3/index.html. Batch query is supported.