The study aims to explore the active molecules of traditional Chinese medicine that specifically bind to interleukin-15 receptor α (IL-15Rα) using molecular docking and surface plasmon resonance (SPR) technology. AutoDock molecular docking software was used to perform simulated docking of more than 3 000 compounds from 48 traditional Chinese medicines at IL-15Rα and screen the specific binding compounds. Then Biocore T200 biomolecular interaction analysis system of SPR was used to confirm the binding specificity of the selected target compounds. Finally, the biological effects of the target compounds on IL-15Rα were verified by cell biological experiments. The results showed that neoprzewaquinone A (Neo) possessed the highest specific binding affinity among the active molecules from traditional Chinese medicine, and the dissociation constant (K_D) value was (0.62 ± 0.20) µmol/L. The results of cell experiment showed that Neo significantly inhibited the proliferation of Mo7e cells induced by IL-15, and the IC₅₀ was 1.075 µmol/L, approximately 1/120 of the IC₅₀ of Cefazolin (IL-15 specific antagonist). These results suggest that Neo is a specific inhibitor of IL-15Rα and may be a potential active drug for the treatment of diseases related to the dysfunction of the IL-15Rα signaling.
Molecular Docking Simulation ; Interleukin-15/pharmacology* ; Surface Plasmon Resonance ; Interleukin-15 Receptor alpha Subunit/metabolism* ; Protein Binding

Imported malaria has become a major risk factor for malaria prevention and control in China. How to screen malaria quickly for people entering China is an urgent problem to be solved. Protein microarrays are widely used in high-throughput screening and diagnosis. In this study, surface plasmon resonance (SPR) technique for malaria detection was established by using the specific adsorption surface treated by polyethylene glycol polymer, and the malaria specific antigen HRP2 was used as capture probe. The optimal concentration of antigen, sensitivity and specificity of detection, as well as anti-interference ability of the chip were analyzed. The SPR protein chip was applied to detect specific antibodies of malignant malaria in serum with the advantage of label-free, instant and fast. Compared with fluorescence quantitative PCR, there were no significant difference in sensitivity and specificity between the two methods. This study lays a foundation for further development of protein microarray for malaria typing identification, and it is conducive to the rapid screening of malaria for people entering.
Antibodies ; China ; Humans ; Malaria/diagnosis* ; Protein Array Analysis ; Surface Plasmon Resonance

OBJECTIVE: To establish quantitative surface plasmon resonance (SPR) assay for antibodies against human platelet antigen-1a (HPA-1a). METHODS: Recombinant protein was fixed on the chip surface by amino coupling method. SPR assay was used to detect the standard antibodies against HPA-1a at different conceatration. The optimal experimental parameters were determined, and standard curves were constructed with linear regression. Moreover, the sensitivity, specificity, accuracy and precision of the assay were evaluated. RESULTS: The quantitative SPR assay for HPA-1a antibodies was established. The determination ranges were 0-20 IU, with accuracy (recovery rate) was 97.75%-103.08%. The intra-assay precision [coefficients of variation (CV)] was 3.53%-4.29%, and the inter-assay precision (CV) was 2.08%-4.40%. For specificity test, several kinds of monoclonal and human antibodies against platelet membrane protein were tested and no positive result was observed. CONCLUSION The established quantitative SPR assay for HPA-1a antibodies shows good sensitivity, specificity, accuracy and precision, and this rapid and simple method provides a new reference method for scientific research and clinical antibody detection.
Antigens, Human Platelet ; Blood Platelets ; Humans ; Isoantibodies ; Surface Plasmon Resonance

Background and Objectives: Endemic plants are integral part of the ecosystem that provide innumerable benefits. Thus, emerging studies and applications using these endemic plants continue to increase globally. In this study, the preparation of gold nanoparticles (AuNPs) has been carried out by using the aqueous leaves extract of Dillenia philippinensis Rolfe. (Katmon) as the bioreductants. Methodology: After the synthesis of AuNPs, the process was further subjected to optimization to assure the production of AuNPs with the best parameters. Synthesized and optimized AuNPs were characterized using FTIR, XRD, DLS, Zeta Potential, and TEM. Results: Optimization results that provide the desired properties for AuNPs were a volume ratio of 1:2 (HAuCl₄: leaves extract), no significant difference (p>0.05) with the sequence of addition and reaction time, and acidic pH. The synthesized AuNPs' particles were found to contain hydroxyl and amine groups, had broad, amorphous, and spherical particles that have a mean diameter of 60.6nm, a PDI of 0.563, and a repulsion force of -13.720mV. The optimized and characterized AuNPs were then further used as a colorimetric sensing material, showing the potential applicability of AuNPs in the heavy metal analysis. Conclusion Gold nanoparticles were able to be synthesized with the use of bioreductants that are present in the aqueous leaves extract of Katmon. This concludes that endemic plants in the Philippines can be used for the synthesis of AuNPs and can be applied in the field of phytonanotechnology and other applied sciences.
Surface Plasmon Resonance

OBJECTIVE: To investigate the feasibilily of screening and identifying the red blood cell type alloantibodies by means of surface plasman resonance(SPR) technique so as to provide a new method for detecting the transfusion compatibility of red blood cells. METHODS: The RBC antigens for screening the alloantibody were fixed on the SPR chip surface by means of amino coupling method; the analysis conditions of SPR chip were optimized and then the control serum with RBC blood group antibody positive was detected; the performance of SPR chip for detection of serum was analysed; the consistance of rusults detected by SPR technique and microcolum agglutination for clinieal samples of 129 thalasstmia patients with history of lone-term blood transfusion were compared; at the same time, the blood group amtibodies in 7 patients with blood group antibody positive were identified before blood transfusion by using SPR chip so as to select the RBC antigen compatible blood for transfusion; and the efficacy of RBC transfusion was followed up and evaluated. RESULTS: The repeatability, sensitivity and specificity of SPR chip technique for detecting the blood group alloantibodies all were better. The SPR technique and microcolumn agglutination method were not significant different for screening blood group alloantibodies (χ2 = 0.333, P＞0.05), and the overall consistency was 97.2%; the results of SPR technique in 7 patients with positive blood group antibodies were as follows: 3 cases with anti-E, 1 case anti-M, 1 case anti-C, 1 case anti-Jka and 1 case autoantibody, which were consistent with the results of microcolumn agglutination tests, and the compatible red blood cells were selected for transfusion, of which the infusion of 6 cases was effective. In only 1 case the infusion was ineffective because of autoantibody. CONCLUSION For screening and identification of blood group alloantibodies, the performance of SPR chip technique is equivalent to the micro-column agglutination, but the procedure of SPR technique is simpler, faster and high-throughput and label-free, which can meet the basic requirements for rapid screening and identification of blood group alloantibodies before transfusion of red blood cells.
Blood Group Antigens ; Blood Transfusion ; Erythrocytes ; Humans ; Isoantibodies ; Surface Plasmon Resonance

PURPOSE: The p38 mitogen-activated protein kinase (MAPKs) play a crucial role in the production of pro-inflammatory cytokines and over-expression of it increase cytokines which promote cancer. Among four isoforms, p38α has been well studied in head and neck squamous cell carcinoma (HNSCC) and other cancers as a therapeutic target. p38δ has recently emerged as a potential disease-specific drug target. Elevated serum p38α level in HNSCC was reported earlier from our lab. This study aims to estimate the levels of p38 MAPK-isoforms in the serum of HNSCC and design peptide inhibitor targeting the same. MATERIALS AND METHODS: Levels of p38 MAPK isoforms in the serum of HNSCC and healthy controls were quantified by surface plasmon resonance technology. The peptide inhibitor for p38 MAPK was designed by molecular modeling using Grid-based Ligand Docking with Energetics tools and compared with known specific inhibitors. RESULTS: We have observed highly elevated levels of all four isoforms of p38 MAPK in serum of HNSCC patients compared to the control group. Further, serum p38α, p38β, and p38δ levels were down regulated after therapy in follow-up patients, while p38γ showed no response to the therapy. Present study screened designed peptide WFYH as a specific inhibitor against p38δ. The specific inhibitor of p38δ was found to have no effect on p38α due to great structural difference at ATP binding pocket. CONCLUSION: In this study, first time estimated the levels of p38 MAPK isoforms in the serum of HNSCC. It can be concluded that p38 MAPK isoforms can be a diagnostic and prognostic marker for HNSCC and p38δ as a therapeutic target.
Adenosine Triphosphate ; Carcinoma, Squamous Cell* ; Cytokines ; Epithelial Cells* ; Follow-Up Studies ; Head* ; Humans ; Models, Molecular ; Neck* ; p38 Mitogen-Activated Protein Kinases ; Protein Isoforms* ; Protein Kinases ; Surface Plasmon Resonance

Reslizumab and mepolizumab are recently approved monoclonal antibodies for the treatment of severe (uncontrolled) eosinophilic asthma. Both are effective in neutralizing the function of interleukin-5 (IL-5). This study is the first to compare the binding affinity and in vitro potency of both antibodies in head-to-head assays. Two assays assessed binding affinity (using the equilibrium dissociation constant [K(D)]) of each drug for human IL-5. In the Biacore surface plasmon resonance assay, the association constant (k(on)) values for human IL-5 for reslizumab and mepolizumab were 3.93 × 10⁶ and 1.83 × 10⁵, respectively. The dissociation constant (k(off)) values were 4.29 × 10⁻⁴ and 2.14 × 10⁻⁴, respectively. Calculated K(D) values for human IL-5 for reslizumab and mepolizumab were 109 and 1,170 pM, respectively, representing an approximately 11-fold stronger binding affinity with reslizumab. In the Kinetic Exclusion Assay, the k(on) values for human IL-5 for reslizumab and mepolizumab were 3.17 × 10⁶ and 1.32 × 10⁵, respectively. The k(off) values were 1.36 × 10⁻⁵ and 1.48 × 10⁻⁵, respectively. Measured K(D) values for human IL-5 for reslizumab and mepolizumab were 4.3 and 112 pM, respectively, representing an approximately 26-fold stronger binding affinity for reslizumab. A human-IL-5-dependent cell proliferation assay was developed to assess in vitro potency, based on a human cell line selected for enhanced surface expression of IL-5 receptor-alpha and consistent proliferation response to IL-5. The concentration at which 50% inhibition occurred (IC₅₀) was determined for both antibodies. Reslizumab and mepolizumab inhibited IL-5-dependent cell proliferation, with IC₅₀ values of approximately 91.1 and 286.5 pM, respectively, representing on average 3.1-fold higher potency with reslizumab. In conclusion, comparative assays show that reslizumab has higher affinity binding for and in vitro potency against human IL-5 compared with mepolizumab. However, these results do not take into consideration the different methods of administration of reslizumab and mepolizumab.
Antibodies ; Antibodies, Monoclonal ; Antibody Affinity ; Asthma ; Cell Line ; Cell Proliferation ; Drug Evaluation, Preclinical ; Eosinophils ; Humans ; In Vitro Techniques ; Interleukin-5 ; Surface Plasmon Resonance

BACKGROUNDCryopyrin-associated periodic syndrome (CAPS) is a group of rare, heterogeneous autoinflammatory disease characterized by interleukin (IL)-1β-mediated systemic inflammation and clinical symptoms involving skin, joints, central nervous system, and eyes. It encompasses a spectrum of three clinically overlapping autoinflammatory syndromes including familial cold autoinflammatory syndrome, Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease. CAPS is associated with gain-of-function missense mutations in NOD-like receptor family pyrin domain-containing protein 3 (NLRP3), the gene encoding NLRP3. Moreover, most mutations leading to MWS occurred in exon 3 of NLRP3 gene. Here, we reported a novel mutation occurred in exon 1 of NLRP3 gene in an MWS patient and attempted to explore the pathogenic mechanism.

METHODSGenetic sequence analysis of NLRP3 was performed in an MWS patient who presented with periodic fever, arthralgia, and multiform skin lesions. NLRP3 was also analyzed in this patient's parents and 50 healthy individuals. Clinical examinations including X-ray examination, skin biopsy, bone marrow aspiration smear, and blood test of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), serum levels of IL-1β, immunoglobulin E (IgE), antineutrophil cytoplasmic antibodies, antinuclear antibodies, and extractable nuclear antigen were also analyzed. The protein structure of mutant NLRP3 inflammasome was calculated by SWISS-MODEL software. Proteins of wild type and mutant components of NLRP3 inflammasome were expressed and purified, and the interaction abilities between these proteins were tested by surface plasmon resonance (SPR) assay.

RESULTSX-ray examination showed no abnormality in the patient's knees. Laboratory tests indicated an elevation of CRP (233.24 mg/L) and ESR (67 mm/h) when the patient had fever. Serum IL-1β increased to 24.37 pg/ml, and serum IgE was higher than 2500.00 IU/ml. Other blood tests were normal. Bone marrow aspiration smear was normal. A novel point mutation c.92A>T in exon 1 of NLRP3 gene was identified, which caused a p.D31V mutation in pyrin domain (PYD) of NLRP3. SPR assay showed that this point mutation may strengthen the interaction between the PYD of NLRP3 and the PYD of the apoptosis-associated speck-like protein. The mutation c.92A>T in exon 1 of the NLRP3 gene was not found in the patient's parents and 50 healthy individuals.

CONCLUSIONSThe mutation c.92A>T in exon 1 of the NLRP3 gene is a novel mutation associated with MWS. The p.D31V mutation might promote the activation of NLRP3 inflammasome and induce MWS in this patient.

Adolescent ; Cryopyrin-Associated Periodic Syndromes ; genetics ; metabolism ; Exons ; genetics ; Humans ; Immunoglobulin E ; blood ; Interleukin-1beta ; blood ; Male ; Mutation ; genetics ; NLR Family, Pyrin Domain-Containing 3 Protein ; genetics ; Surface Plasmon Resonance

PURPOSE: Dickkopf 1 (DKK1) has been extensively investigated in mouse models of multiple myeloma, which results in osteolytic bone lesions. Elevated DKK1 levels in bone marrow plasma and serum inhibit the differentiation of osteoblast precursors. Present pharmaceutical approaches to target bone lesions are limited to antiresorptive agents. In this study, we developed a cyclized oligopeptide against DKK1-low density lipoprotein receptor-related protein (LRP) 5/6 interaction and tested the effects of the oligopeptide on tumor burden. MATERIALS AND METHODS: A cyclized oligopeptide based on DKK1-LRP5/6 interactions was synthesized chemically, and its nuclear magnetic resonance structure was assessed. Luciferase reporter assay and mRNA expressions of osteoblast markers were evaluated after oligopeptide treatment. MOPC315.BM.Luc cells were injected into the tail vein of mice, after which cyclized oligopeptide was delivered subcutaneously 6 days a week for 4 weeks. RESULTS: The cyclized oligopeptide containing NXI motif bound to the E1 domain of LRP5/6 effectively on surface plasmon resonance analysis. It abrogated the Wnt-β-catenin signaling inhibited by DKK1, but not by sclerostin, dose dependently. RT-PCR and alkaline phosphatase staining showed increased expressions of osteoblast markers according to the treatment concentrations. Bioluminescence images showed that the treatment of cyclized oligopeptide reduced tumor burden more in oligopeptide treated group than in the vehicle group. CONCLUSION: The cyclized oligopeptide reported here may be another option for the treatment of tumor burden in multiple myeloma.
Alkaline Phosphatase ; Animals ; Bone Density Conservation Agents ; Bone Marrow ; Lipoproteins ; Luciferases ; Magnetic Resonance Spectroscopy ; Mice* ; Multiple Myeloma* ; Osteoblasts ; Plasma ; RNA, Messenger ; Surface Plasmon Resonance ; Tail ; Tumor Burden* ; Veins

Gene expression exhibits temporal and spatial patterns to response environmental changes and growth cycle. Gene expression is under strict control at different levels among which control at transcription level is the predominant mode, especially in prokaryotes. In this review, we summarized the new developments of methods used in transcriptional studies, including modifications and improvements of the classic methods, such as gel-shift assay, DNA foot printing, and in vivo reporter system. In addition, we introduced examples to apply new methods, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize protein-DNA, ligand-protein, and ligand-protein-DNA interactions. The collection of these methods and their application could guide and accelerate relevant studies.
Calorimetry ; DNA Footprinting ; Gene Expression ; Ligands ; Proteins ; Surface Plasmon Resonance ; Transcription, Genetic