Structure-based improvement of the binding affinity and recognition specificity of peptide competitors to target pediatric IL-5R/IL-5 interaction by gluing halogen bonds at their complex interface.

Human interleukin-5 (IL-5) cytokine mediates the development of eosinophils and is involved in a variety of immune inflammatory responses that play a major role in the pathogenesis of childhood asthma, leukemia, and other pediatric allergic diseases. The immunomodulatory cytokine functions by binding to its cognate cell surface receptor IL-5R in a sheet-by-sheet manner, which can be conformationally mimicked and competitively disrupted by a double-stranded cyclic AF18748 peptide. In this study, we systematically examined the co-crystallized complex structure of human IL-5R with AF18748 peptide and rationally designed a halogen bond to glue at the protein-peptide complex interface by substituting the indole moiety of AF18748 Trp13 residue with a halogen atom (X = F, Cl, Br, or I). High-level theoretical calculations imparted presence of the halogen bond between the oxygen atom (O) of IL-5R Glu58 backbone and the halogen atom (X) of AF18748 Trp13 side chain. Experimental assays confirmed that the halogen bond can promote peptide binding moderately or considerably. More importantly, the halogen bond not only enhances peptide affinity to IL-5R, but also improves peptide selectivity for its cognate IL-5R over other noncognate IL-R proteins. As might be expected, the affinity and selectivity conferred by halogen bond increase consistently in the order: H < F < Cl < Br < I. Structural modeling revealed that the halogen bond plus its vicinal π-cation-π stacking co-define a ringed noncovalent system at the complex interface, which involves a synergistic effect to effectively improve the peptide binding potency and recognition specificity.

Extracellular vesicles derived from miR-199a-5p-modified adipose-derived mesenchymal stem cells alleviate immune thrombocytopenia by inhibiting T helper 17 differentiation.

The abnormal differentiation of T helper 17 (Th17) cells is considered a vital promoter of immune thrombocytopenia (ITP) progression. Therefore, this study investigated the role of miR-199a-5p in Th17 differentiation and determined whether extracellular vesicles (EVs) derived from miR-199a-5p-modified adipose-derived mesenchymal stem cells (ADSCs) could relieve ITP by inhibiting Th17 differentiation. The miR-199a-5p level was lessened in the spleen tissues of mice with ITP, while the signal transducer and activator of transcription 3 (STAT3) expression and the population of Th17 in CD4+T cells were boosted. Functionally, miR-199a-5p overexpression lowered IL-17 secretion and the proportion of Th17/CD4+T cells. Further investigation showed that miR-199a-5p directly targeted STAT3 mRNA, and negatively modulated its expression. STAT3 overexpression was found to facilitate Th17 differentiation, which was subsequently abolished by miR-199a-5p overexpression. EVs isolated from miR-199a-5p-modified ADSCs (miR-199a-5p-EVs) highly expressed miR-199a-5p and could restrain CD4+T cells polarized toward a Th17 phenotype in vitro. Administering of miR-199a-5p-EVs elevated platelet counts and decreased the proportion of Th17/CD4+T cells in mice with ITP. Taken together, EVs derived from miR-199a-5p-modified ADSCs vividly repressed Th17 differentiation by transferring miR-199a-5p to CD4+T cells, thus ameliorating experimental ITP.

HUWE1 Causes an Immune Imbalance in Immune Thrombocytopenic Purpura by Reducing the Number and Function of Treg Cells Through the Ubiquitination Degradation of Ets-1.

Background: Immune thrombocytopenic purpura (ITP) is an autoimmune bleeding disorder and the decreased number and immunosuppressive dysfunction of Treg cells are key promoters of ITP. However, their mechanisms in ITP development have not been fully clarified. Methods: HUWE1 mRNA and protein levels in CD4+ T cells in peripheral blood from ITP patients were assessed by quantitative real-time PCR and Western blot. HUWE1 function in ITP was estimated using flow cytometry, enzyme-linked immunosorbent assay and immunosuppression assay. Besides, the HUWE1 mechanism in reducing the number and function of Treg cells in ITP was investigated by immunoprecipitation, cycloheximide-chase assay, ubiquitin experiment and immunofluorescence assay. Results: HUWE1 expression was elevated in CD4+ T cells in peripheral blood from ITP patients and HUWE1 mRNA level was negatively correlated with platelet counts and Treg cell percentage. Moreover, the interference with HUWE1 increased the number of Treg cells and enhanced its immunosuppressive function, and the HUWE1 overexpression produced the opposite results. For the exploration of mechanism, HUWE1 interacted with E26 transformation-specific-1 (Ets-1) and this binding was dependent on the negative regulation of the phosphorylation level of Ets-1 (Thr38) and HUWE1 facilitated the ubiquitin degradation of Ets-1 protein to restrain Treg cell differentiation and weaken their immunosuppressive functions. The in vivo assay confirmed that the HUWE1 inhibitor alleviated ITP in mice. Conclusion: HUWE1 induced the immune imbalance in ITP by decreasing the number and weakening the function of Treg cells through the ubiquitination degradation of Ets-1.

LncRNA GAS5 inhibits Th17 differentiation and alleviates immune thrombocytopenia via promoting the ubiquitination of STAT3.

BACKGROUND: The increased differentiation of T helper 17 cells (Th17) accelerates the development of immune thrombocytopenia (ITP), which is a common autoimmune disease with limited therapeutic methods. Recent studies have revealed that long non-coding RNAs (lncRNAs) play a critical role in autoimmune diseases, thus this study aims to investigate the effect of lncRNA GAS5 on the differentiation of Th17 cells in ITP. METHODS: The expression of GAS5 in peripheral blood mononuclear cells (PBMCs) of ITP patients and spleen tissues of ITP mice was measured by qRT-PCR. The percentage of Th17 cells in CD4+ cells was measured by flow cytometry. The combination between GAS5 and STAT3 was confirmed by RNA pull-down assay and RNA Binding Protein Immunoprecipitation (RIP). The ubiquitination of STAT3 was detected by ubiquitination assay and the interaction between STAT3 and TRAF6 was measured by Co-Immunoprecipitation (Co-IP). Finally, the effect of GAS5 on Th17 differentiation was investigated in vitro and in vivo using lentivirus (lenti)-GAS5. RESULTS: GAS5 expression was downregulated both in PBMCs of ITP patients and spleen tissues of ITP mice. Overexpression of GAS5 suppressed Th17 differentiation while had no effect on Treg differentiation in naïve CD4+ cells. RNA pull-down and RNA immunoprecipitation assays confirmed the interaction between GAS5 and STAT3. Further studies showed GAS5 accelerated the degradation of STAT3 via promoting TRAF6-mediated ubiquitination. Overexpressing GAS5 suppressed Th17 differentiation in vitro and alleviated ITP in vivo via reducing STAT3. CONCLUSION: LncRNA GAS5 inhibited Th17 differentiation through promoting the TRAF6-mediated ubiquitination of STAT3, thus relieving ITP.

miR-106b-5p induces immune imbalance of Treg/Th17 in immune thrombocytopenic purpura through NR4A3/Foxp3 pathway.

BACKGROUND: Immune imbalance of regulatory T cells (Treg)/T helper 17 cells (Th17) contributes to the development of immune thrombocytopenic purpura (ITP). The dysregulation of miRNAs is important in the development of ITP. However, the role of miR-106b-5p in Treg/Th17 imbalance remains unknown in ITP. MATERIALS AND METHODS: Peripheral blood was collected from patients with ITP and healthy controls, and CD4 + T cells were further isolated. miR-106b-5p, nuclear receptor subfamily 4 group A member 3 (NR4A3), forkhead box protein 3 (Foxp3), IL-17A, and TGF-ß expressions were detected by qRT-PCR, western blot, or ELISA. The effect of miR-106b-5p on NR4A3 was detected by dual-luciferase reporter gene assay. RESULTS: Compared with healthy controls, miR-106b-5p was elevated in peripheral blood of patients with ITP, and NR4A3 expression was decreased. sh-NR4A3 significantly decreased Foxp3 and TGF-ß expressions, indicating that NR4A3 may regulate Treg differentiation via Foxp3. Additionally, NR4A3 was identified to be a target of miR-106b-5p, and miR-106b-5p was able to negatively modulate NR4A3 expression. Moreover, we found miR-106b-5p induced immune imbalance of Treg/Th17 through NR4A3. In vivo experiments revealed that silencing miR-106b-5p promoted Treg differentiation and increased the number of platelets, suggesting the relief of ITP. CONCLUSION: miR-106b-5p regulated immune imbalance of Treg/Th17 in ITP through the NR4A3/Foxp3 pathway.

A novel and label-free biosensors for uracil-DNA glycosylase activity based on the electrochemical oxidation of guanine bases at the graphene modified electrode.

Uracil-DNA glycosylase (UDG) as an important base excision repair enzymes is widely distributed in organism, and it plays a crucial role in sustaining the genome integrity. Therefore, it is significant to carry out the analysis of UDG activity. In this present work, a novel and label-free electrochemical sensing platform for the sensitive detection of uracil DNA glycosylase (UDG) activity has been developed. Herein, the graphene modified glassy carbon (GC) electrode was prepared. And two complementary DNA strands were hybridized to form dsDNA (P1P2). In the presence of UDG, the uracil bases in P1P2 were specifically hydrolyzed, inducing the unwinding of the DNA duplex, and accompanied by the release of P1. Thus, the released P1 was adsorbed onto the graphene/GC electrode surface via π-π stacking. By investigating the electrochemical behavior of P1 at the graphene/GC electrode, the electrochemical oxidation of guanine bases in P1 was obviously observed. Therefore, using the current responses of guanine base in P1 as a signal indicator, UDG activity can be simply determined with high sensitivity, and the detectable lowest concentration is 0.01U/mL. This present design does not need covalent attachment of redox indicator to DNA, preventing participation of redox labels in the background. Meanwhile, the proposed strategy for the assay of UDG activity also has a remarkable sensitivity due to the excellent properties of graphene, which could increase both the immobilization amount of released ssDNA and the conductivity of the sensing system. All these elucidate that this developed protocol may lay a potential foundation for the sensitive detection of UDG activity in clinical diagnosis.

A sensitive enzymeless sensor for hydrogen peroxide based on the polynucleotide-templated silver nanoclusters/graphene modified electrode.

A novel, sensitive and enzymeless electrochemical sensor based on polynucleotide-templated silver nanoclusters (DNA-AgNCs)/graphene composite film was developed for the detection of hydrogen peroxide. The graphene modified glassy carbon electrode (GCE) was employed because graphene has several advantages including excellent conductivity, biocompatibility, and large surface area to volume ratio. In addition, it was found that DNA-AgNCs have remarkable electrocatalytic activity toward the reduction of hydrogen peroxide, and can be easily immobilized onto the surface of the graphene/GCE by π-π stacking. The sensor based on the (DNA-AgNCs)/graphene/GCE exhibited a rapid response (ca. 3s), a low detection limit (3 µM), a wide linear range from 15 µM to 23 mM, high selectivity, as well as good repeatability. Moreover, the common interfering species, such as ascorbic acid, uric acid, dopamine, glutathione, and l-cysteine, did not result in any interference. This present work may expand the use of silver nanoclusters in the field of electrochemical sensor.

A fluorometric assay for acetylcholinesterase activity and inhibitor detection based on DNA-templated copper/silver nanoclusters.

A novel label-free, rapid, cost-effective, and highly sensitive fluorometric sensor has been constructed for the detection of acetylcholinesterase (AChE) activity and its inhibitor based on the fluorescence quenching of DNA-templated copper/silver nanoclusters (DNA-Cu/AgNCs). In this assay, AChE catalyzes the hydrolysis of acetylthiocholine (ATCh) to form thiocholine which induces fluorescence quenching of DNA-Cu/AgNCs. The AChE activity could be detected as low as 0.05mU/mL and with a linear range from 0.05 to 2.0mU/mL. This assay offers a very convenient "mix and detect" approach for AChE activity. On the other hand, tacrine and organophosphorus pesticides (OPPs) were employed to inhibit the hydrolysis of ATCh, which could eliminate the fluorescence quenching of DNA-Cu/AgNCs. The IC50 of tacrine and methamidophos were estimated to be 16.9nM and 0.075mg/L, respectively. This method was also used to detect spiked OPPs in agricultural products successfully. The present work may expand the use of DNA-Cu/AgNCs to the field of enzyme sensors.

Electrochemical oxidation of purine and pyrimidine bases based on the boron-doped nanotubes modified electrode.

Based on the excellent physicochemical properties of boron-doped carbon nanotubes (BCNTs), the electrochemical analysis of four free DNA bases at the BCNTs modified glassy carbon (GC) electrode was investigated. Herein, the BCNTs/GC electrode exhibited remarkable electrocatalytic activity towards the oxidation of purine bases (guanine (G), adenine (A)). More significantly, the direct oxidation of pyrimidine bases (thymine (T), cytosine (C)) was realized. It may be due to that BCNTs have the advantages of high electron transfer kinetics, large surface area, prominent antifouling ability and electrode activity. On basis of this, a novel and simple strategy for the determination of G, A, T and C was proposed. The BCNTs/GC electrode showed high sensitivity, wide linear range and capability of detection for the electrochemical determination of G, A, T, and C. On the other hand, the electrochemical oxidation of quaternary mixture of G, A, T, and C at the BCNTs/GC electrode was investigated. It was obtained that the peak separation between G and A, A and T, T and C were large enough for their potential recognition in mixture without any separation or pretreatment. The BCNTs/GC electrode also displayed good stability, reproducibility and excellent anti-interferent ability. Therefore, it can be believed that the BCNTs/GC electrode would provide a potential application for the electrochemical detection of DNA in the field of genetic-disease diagnosis.