Medium-high resolution electrochemical genotyping of HLA-DQ2/DQ8 for detection of predisposition to coeliac disease

Coeliac disease is a small intestinal disorder, induced by ingestion of gluten in genetically predisposed individuals. Coeliac disease has been strongly linked to human leukocyte antigens (HLA) located on chromosome 6, with almost 100 % of coeliac disease sufferers carrying either a HLA-DQ2 or HLA-DQ8 heterodimer, with the majority carrying HLA-DQ2 encoded by the DQA1*05:01/05:05, DQB1*02:01/02:02 alleles, whereas the remaining carry the HLA-DQ8 encoded by the DQA1*03:01, DQB1*03:02 alleles. In this work, we present the development of a multiplex electrochemical genosensor array of 36 electrodes, housed within a dedicated microfluidic platform and using a total of 10 sequence-specific probes for rapid medium-high resolution HLA-DQ2/DQ8 genotyping. An evaluation of the selectivity of the designed probes was carried out with the target sequences and 44 potentially interfering alleles, including single base mismatch differentiations; good selectivity was demonstrated. The performance of the electrochemical genosensor array was validated, analyzing real human samples for the presence of HLA-DQ2/DQ8 alleles, and compared with those obtained using laboratory-based HLA typing, and an excellent correlation was obtained.

Inhibition of HLA-DQ2-mediated antigen presentation by analogues of a high affinity 33-residue peptide from alpha2-gliadin

Human leukocyte antigen DQ2 is a class II major histocompatibility complex protein that plays a critical role in the pathogenesis of Celiac Sprue by binding to epitopes derived from dietary gluten and triggering the inflammatory response of disease-specific T cells. Inhibition of DQ2-mediated antigen presentation in the small intestinal mucosa of Celiac Sprue patients therefore represents a potentially attractive mode of therapy for this widespread but unmet medical need. Starting from a pro-inflammatory, proteolytically resistant, 33-residue peptide, LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF, we embarked upon a systematic effort to dissect the relationships between peptide structure and DQ2 affinity and to translate these insights into prototypical DQ2 blocking agents. Three structural determinants within the first 20 residues of this 33-mer peptide, including a PQPELPYPQ epitope, its N-terminal flanking sequence, and a downstream Glu residue, were found to be important for DQ2 binding. Guided by the X-ray crystal structure of DQ2, the L11 and L18 residues in the truncated 20-mer analogue were replaced with sterically bulky groups so as to retain high DQ2 affinity but abrogate T cell recognition. A dimeric ligand, synthesized by regiospecific coupling of the 20-mer peptide with a bifunctional linker, was identified as an especially potent DQ2 binding agent. Two such ligands were able to attenuate the proliferation of disease-specific T cell lines in response to gluten antigens and, therefore, represent prototypical examples of pharmacologically suitable DQ2 blocking agents for the potential treatment of Celiac Sprue.     

In silico epitope identification of unique multidrug resistance proteins from Salmonella Typhi for vaccine development

Typhoid fever is a multisystemic illness caused by Salmonella enterica serovars Typhi and is resistant to most antibiotics and drugs. The resistance is conferred through multidrug resistance (MDR) proteins, which efflux most antibiotics and other drugs. We predicted potential candidate B-cell and T-cell epitopes using bio- and immune-informatics tools in the 11 MDR proteins - EmrA, EmrB, EmrD, MdtA, MdtB, MdtC, MdtG, MdtH, MdtK, MdtL and TolC. The antigenic potential of the MDR proteins was calculated using VaxiJen server. The B-cell and T-cell epitopes of the MDR proteins were predicted using BCPred and ProPredI and ProPred respectively. The binding affinities of the predicted T-cell epitopes were estimated using T-epitope designer and MHCPred tools. 10, 7, 5, 12, 14, 21, 26, 3, 3 and 3 B-cell epitopes were identified in EmrA, EmrB, EmrD, TolC, MdtA, MdtB, MdtC, MdtG, MdtH and MdtL respectively. We predicted 9 T-cell epitopes - YVSRRAVQP (EmrA), FGVANAISI (EmrB), MVNSQVKQA and YQGGMVNSQ (TolC), WDRTNSHKL (MdtA), FLRNIPTAI (MdtB), YVEQLGVTG (MdtG), VKWMYAIEA (MdtH) and LAHTNTVTL (MdtL) capable of eliciting both humoral and adaptive immune responses. These T-cell epitopes specifically bind to HLA alleles - DRB1*0101 and DRB1*0401. This is the first report of epitope prediction in the MDR proteins of S. Typhi. Taken together, these results indicate the MDR proteins – EmrA, MdtA and TolC are the most suitable vaccine candidates for S. Typhi. The findings of our study on the MDR proteins prove to be useful in the development of peptide-based vaccine for the prevention and/or treatment of typhoid fever.     

Resolving Multiple Protein–Peptide Binding Events: Implication for HLA-DQ2 Mediated Antigen Presentation in Celiac Disease

Techniques that can effectively separate protein–peptide complexes from free peptides have shown great value in major histocompatibility complex (MHC)–peptide binding studies. However, most of the available techniques are limited to measuring the binding of a single peptide to an MHC molecule. As antigen presentation in vivo involves both endogenous ligands and exogenous antigens, the deconvolution of multiple binding events necessitates the implementation of a more powerful technique. Here we show that capillary electrophoresis coupled to fluorescence detection (CE–FL) can resolve multiple MHC–peptide binding events owing to its superior resolution and the ability to simultaneously monitor multiple emission channels. We utilized CE–FL to investigate competition and displacement of endogenous peptides by an immunogenic gluten peptide for binding to HLA-DQ2. Remarkably, this immunogenic peptide could displace CLIP peptides from the DQ2 binding site at neutral but not acidic pH. This unusual ability of the gluten peptide supports a direct loading mechanism of antigen presentation in extracellular environment, a property that could explain the antigenicity of dietary gluten in celiac disease.     

Conformational study of the complementary peptide to a B-cell epitope of the La/SSB autoantigen

Starting from the 20-mer peptide 289–308, one of the experimentally characterized B-cell epitopes of the La/SSB autoantigen, the complementary peptide cpl(289–308), encoded by the complementary RNA was designed. The conformational properties of the cpl(289–308) were investigated in DMSO solution with the combined use of NMR data (vicinal coupling constants, NOE effects and temperature coefficient values), molecular modelling calculations of energy minimization and molecular dynamics. MD calculations led to a folded structure in which a βI-turn, stabilized by the H₈ amide proton to the F₅ carbonyl hydrogen bond, was found for the F₅P₆S₇H₈ sequence, whereas two γ-turns, centred around the E₁₅ and I₁₈ residues respectively, were found in the C-terminal part of the peptide. In the whole crown folded structure of the peptide, the Y₄, F₅, H₈, F₉ and F₁₀ aromatic side chains are situated on one side with the E₁₃, E₁₅, T₁₇ and C₂₀ side chains on the other. This 3D structure resembles and could mimic the binding site of an antibody.     

Homonuclear SQ-DQ correlations in solids: applications of the broadband BaBa scheme to reveal 31P-31P spatial correlations in two-, three-, four-, and five-spin systems

In this paper, we report on the use and limitations of the popular double‐quantum recoupling sequence back‐to‐back in studies of ³¹P‐³¹P spatial proximities in Pd‐phosphine complexes at medium field (9.38 T) under the conditions of fast MAS. The effects of internuclear distances in different spin systems and the impact of isotropic chemical shift and chemical shift anisotropy (CSA) offsets on the detectibility of SQ‐DQ correlations were of particular interest to us. Selected model compounds with these requirements in mind were synthesized. By optimization of the excitation times of SQ‐DQ correlations up to 4.6 Å could be obtained even in four‐spin systems; however, certain long‐range correlations may be weak or missing. Although under fast MAS, CSA values up to 270 ppm are well tolerated, in multi‐spin systems in cases of isotropic chemical shift offsets larger than 50 ppm, the DQ coherences cannot be properly excited. Copyright © 2011 John Wiley & Sons, Ltd.     

Low-medium resolution HLA-DQ2/DQ8 typing for coeliac disease predisposition analysis by colorimetric assay

Coeliac disease is an inflammation of the small intestine, occurring in genetically susceptible individuals triggered by the ingestion of gluten. Human Leukocyte Antigens (HLA) DQ2 and DQ8 gene have been identified as key genetic factors in coeliac disease as they are presented in almost 100 % of the patients. These genes are encoded by the combination of certain alleles in the DQA and DQB region of chromosome 6. Specifically, DQA1*05:01 and DQB1*02:01 alleles for serologically defined leukocyte antigen DQ2 cis, DQA1*05:05 and DQB1*02:02 for DQ2 trans and DQA1*03:01 and DQB1*03:02 alleles for the DQ8. Specific identification of these alleles is a challenge due to the high number of alleles that have been identified so far: 46 in the DQA region and 160 in the DQB region (as of IMGT/HLA Database 10/2011 release). In the reported work, the development of a multiplex colorimetric assay for the low to medium HLA typing of the DQ2 and DQ8 genes is presented. The optimisation of probe design and assay conditions, performed by both surface plasmon resonance and enzyme-linked oligonucleotide assay, are reported. Finally, the performances of the developed typing platform were validated by the analysis of real patient samples and HLA typing, compared with those obtained using hospital based typing technology and an excellent correlation obtained.     

Autoantibodies to thyroglobulin in health and disease

Thyroglobulin (Tg)—a heavily glycosylated, iodinated protein—isa major autoantigen in autoimmune thyroiditis. Tg also induces thyroiditis by immunization of experimental animals. Humans with chronic lymphocytic thyroiditis characteristically produce autoantibodies to thyroglobu lin, but similar autoantibodies are also found in some clinically normal, euthyroid individuals. A comparison of the fine specificity of autoantibodies in humans and in experimentally immunized mice was carried out, based on their ability to inhibit a panel of monoclonal antibodies (MAbs). Patients with autoimmune thyroid disease, as well as normal individuals, produced autoantibodies mainly to the conserved, cross-reactive determinants of thyroglobulin. Patients developed additional autoantibodies to species-restricted epitopes. The determinants recognized by patients with Graves' disease differed in some respects from epitopes recognized by thyroiditis patients or patients with differentiated thyroid carcinoma. Similarly, mice that are genetically susceptible to thyroiditis produced autoantibodies that reacted with the mouse-specific antigenic determinants. Using an autoantibody that reacts with one of the epitopes associated with thyroiditis, a reactive 15-k Da fragment of human Tg—localized at the carboxy end of the molecule—was isolated and sequenced. Iodine plays an important role in the precise specificity of the disease-associated epitope, since T cells from patients with thyroiditis react with iodinated but not noniodinated human thyroglobulin. Addition of iodine to Tg generates new or cryptic epitopes. Use of a selected MAb as a surrogate for the T-cell receptor suggests that a specific iodine-containing epitope is sometimes involved in recognition. Finally, thyroglobulin-reactive autoantibodies exhibit proteolytic activity on thyroglobulin.     

固相萃取净化-超高效液相色谱-高分辨质谱法测定尿液中百草枯和敌草快残留

尿液样品中百草枯(PQ)和敌草快(DQ)的检测是理化检验工作的难点。PQ和DQ具有分子极性大和水溶性好等特点,常规反相色谱柱难以保留;现有文献方法多采用亲水相互作用色谱法(HILIC)进行保留,但文献方法需采用高浓度缓冲盐作为流动相,增加了质谱仪的污染。基于上述问题,研究建立了弱阳离子交换(WCX)固相萃取净化-超高效液相色谱-高分辨质谱法(UPLC-HRMS)快速准确测定尿液样品中PQ和DQ残留的检测方法。尿液样品经混合磷酸盐缓冲液(pH=6.86)稀释和WCX固相萃取净化后,在Syncronis HILIC色谱柱(100 mm×2.1 mm, 1.7 μm)上进行梯度洗脱分离,采用正离子电喷雾离子化模式(ESI⁺)和一级全扫描-数据依赖二级质谱扫描模式(Full mass-ddMS²)进行定量分析。研究通过对色谱条件的不断优化,将HILIC模式下流动相中甲酸铵缓冲盐的浓度降低至10 mmol/L,并系统优化了样品前处理过程中影响PQ和DQ准确性的因素。在最优条件下,PQ和DQ线性关系良好(r²>0.998),在4个加标水平下(1.0、20.0、100.0和200.0 μg/L), PQ和DQ的平均加标回收率分别为85.8%~101%和80.3%~86.9%,精密度(RSD)分别为0.8%~5.1%和0.9%~4.2%。方法的检出限(S/N≥3)和定量限(S/N≥10)分别为0.2 μg/L和0.6 μg/L。将建立的方法用于中毒病人临床治疗过程尿液中DQ含量的跟踪监测。该方法具有快速、简便、灵敏和准确等优点,适用于临床中毒病例尿液样品中PQ和DQ的检测。     

Inclusion complexation of diquat and paraquat by the hosts cucurbit[7]uril and cucurbit[8]uril

The binding interactions in aqueous solution between the dicationic guest diquat (DQ(2+)) and the cucurbit[7]uril (CB7) and cucurbit[8]uril (CB8) hosts were investigated by (1)H NMR, UV/Vis, and fluorescence spectroscopy; mass spectrometry; single-crystal X-ray diffraction; and electrochemical techniques. The binding data were compared with previously reported results for the related paraquat guest (PQ(2+)). DQ(2+) was found to bind poorly (K=350 m(-1)) inside CB7 and more effectively (K=4.8 x 10(4) m(-1)) inside CB8. One-electron reduction led to increased binding affinity with both hosts (K(r)=1 x 10(4) m(-1) with CB7 and K(r)=6 x 10(5) m(-1) for CB8). While (1)H NMR spectroscopic data revealed that DQ(2+) is not fully included by CB7, the crystal structure of the CB8DQ(2+) complex-obtained from single-crystal X-ray diffraction-clearly establishes its inclusion nature. Overall, both diquat and its one-electron reduced radical cation are bound more effectively by CB8 than by CB7. In contrast to this, paraquat exhibits selectivity for CB7, but its radical cation forms a highly stable dimer inside CB8. These differences highlight the pronounced sensitivity of cucurbit[n]uril hosts to guest features such as charge, charge distribution and shape.     

High magnetic field solid‐state NMR analyses by combining MAS,MQ‐MAS,homo‐nuclear and hetero‐nuclear correlation experiments

A general strategy of structural analysis of alumina silicate by combining various solid‐state NMR measurements such as single pulse, multi‐quantum magic angle spinning, double‐quantum homo‐nuclear correlation under magic angle spinning (DQ‐MAS), and cross‐polarization hetero‐nuclear correlation (CP‐HETCOR) was evaluated with the aid of high magnetic field NMR (800 MHz for ¹H Larmor frequency) by using anorthite as a model material. The high magnetic field greatly enhanced resolution of ²⁷Al in single pulse, DQ‐MAS, and even in triple‐quantum magic angle spinning NMR spectra. The spatial proximities through dipolar couplings were probed by the DQ‐MAS methods for homo‐nuclear correlations between both ²⁷Al–²⁷Al and ²⁹Si–²⁹Si and by CP‐HETCOR for hetero‐nuclear correlations between ²⁷Al–²⁹Si in the anorthite framework. By combining various NMR methodologies, we elucidated detailed spatial correlations among various aluminum and silicon species in anorthite that was hard to be determined using conventional analytical methods at low magnetic field. Moreover, the presented approach is applicable to analyze other alumina‐silicate minerals. Copyright © 2012 John Wiley & Sons, Ltd.     

MALDI‐MS detection of noncovalent interactions of single stranded DNA with Escherichia coli single‐stranded DNA‐binding protein

The Escherichia coli single‐stranded DNA binding protein (SSB) selectively binds single‐stranded (ss) DNA and participates in the process of DNA replication, recombination and repair. Different binding modes have previously been observed in SSB?ssDNA complexes, due to the four potential binding sites of SSB. Here, chemical cross‐linking, combined with high‐mass matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS), is used to determine the stoichiometry of the SSB?ssDNA complex. SSB forms a stable homotetramer in solution, but only the monomeric species (m/z 19 100) can be detected with standard MALDI‐MS. With chemical cross‐linking, the quaternary structure of SSB is conserved, and the tetramer (m/z 79 500) was observed. We found that ssDNA also functions as a stabilizer to conserve the quaternary structure of SSB, as evidenced by the detection of a SSB?ssDNA complex at m/z 94 200 even in the absence of chemical cross‐linking. The stability of the SSB?ssDNA complex with MALDI strongly depends on the length and strand of oligonucleotides and the stoichiometry of the SSB?ssDNA complex, which could be attributed to electrostatic interactions that are enhanced in the gas phase. The key factor affecting the stoichiometry of the SSB?ssDNA complex is how ssDNA binds to SSB, rather than the protein‐to‐DNA ratio. This further suggests that detection of the complex by MALDI is a result of specific binding, and not due to non‐specific aggregation in the MALDI plume. Copyright © 2012 John Wiley & Sons, Ltd.     

In silico identification of outer membrane protein (Omp) and subunit vaccine design against pathogenic Vibrio cholerae

Virulence-related outer membrane proteins (Omps) are expressed in bacteria (Gram-negative) such as V. cholerae and are vital to bacterial invasion in to eukaryotic cell and survival within macrophages that could be best candidate for development of vaccine against V. cholerae. Applying in silico approaches, the 3-D model of the Omp was developed using Swiss model server and validated byProSA and Procheck web server. The continuous stretch of amino acid sequences 26 mer: RTRSNSGLLTWGDKQTITLEYGDPAL and 31 mer: FFAGGDNNLRGYGYKSISPQDASGALTGAKY having B-cell binding sites were selected from sequence alignment after B cell epitopes prediction by BCPred and AAP prediction modules of BCPreds. Further, the selected antigenic sequences (having B-cell epitopes) were analyzed for T-cell epitopes (MHC I and MHC II alleles binding sequence) by using ProPred 1 and ProPred respectively. The epitope (9 mer: YKSISPQDA) that binds to both the MHC classes (MHC I and MHC II) and covers maximum MHC alleles were identified. The identified epitopes can be useful in designing comprehensive peptide vaccine development against V. cholerae by inducing optimal immune response.     

Evaluation of potential MHC-I allele-specific epitopes in Zika virus proteins and the effects of mutations on peptide-MHC-I interaction studied using in silico approaches

Zika virus (ZIKV) infection is a global health concern due to its association with microcephaly and neurological complications. The development of a T-cell vaccine is important to combat this disease. In this study, we propose ZIKV major histocompatibility complex I (MHC-I) epitopes based on in silico screening consensus followed by molecular docking, PRODIGY, and molecular dynamics (MD) simulation analyses. The effects of the reported mutations on peptide-MHC-I (pMHC-I) complexes were also evaluated. In general, our data indicate an allele-specific peptide-binding human leukocyte antigen (HLA) and potential epitopes. For HLA-B44, we showed that the absence of acidic residue Glu at P2, due to the loss of the electrostatic interaction with Lys45, has a negative impact on the pMHC-I complex stability and explains the low free energy estimated for the immunodominant peptide E-4 (IGVSNRDFV). Our MD data also suggest the deleterious effects of acidic residue Asp at P1 on the pMHC-I stability of HLA-B8 due to destabilization of the α-helix and β-strand. Free energy estimation further indicated that the mutation from Val to Ala at P9 of peptide E-247 (DAHAKRQTV), which was found exclusively in microcephaly samples, did not reduce HLA-B8 affinity. In contrast, the mutation from Thr to Pro at P2 of the peptide NS5−832 (VTKWTDIPY) decreased the interaction energy, number of intermolecular interactions, and adversely affected its binding mode with HLA-A1. Overall, our findings are important with regard to the design of T-cell peptide vaccines and for understanding how ZIKV escapes recognition by CD8 + T-cells.     

Common semiopen conformations of Mg2+-free Ras, Rho, Rab, Arf, and Ran proteins combined with GDP and their similarity with GEF-bound forms

A computational study was performed on the Mg(2+)-free conformations of the small guanine nucleotide-binding proteins (GNBPs): Ras, Rho, Rab, Arf, and Ran, which were complexed with GDP. Molecular dynamics (MD) simulation was executed for each complex for the duration of 3.0 ns to investigate the effects of Mg(2+) ions on the GNBPs' structure. The results indicated that all Mg(2+)-free GNBPs formed a groove between the switch region and the nucleotide-binding site. In some GNBP families, the release of Mg(2+) was reported to play an important role in binding the guanine nucleotide-exchanging factor (GEF) promoting the GDP/GTP exchange reaction. Interestingly, the grooves, which appeared in the MD simulations, were similar to the grooves experimentally observed in the GNBP-GEF complex. We also calculated the Mg(2+)-bound GNBPs to compare with the Mg(2+)-free forms. No groove was observed in the Mg(2+)-bound GNBPs. These results demonstrated a regulatory role of Mg(2+) ion to prepare a template for the GEF binding. Moreover, the results suggested that the release of Mg(2+) ion lead to the GEF-GNBP binding.     

A combination of epitope prediction and molecular docking allows for good identification of MHC class I restricted T-cell epitopes

In silico identification of T-cell epitopes is emerging as a new methodology for the study of epitope-based vaccines against viruses and cancer. In order to improve accuracy of prediction, we designed a novel approach, using epitope prediction methods in combination with molecular docking techniques, to identify MHC class I restricted T-cell epitopes. Analysis of the HIV-1 p24 protein and influenza virus matrix protein revealed that the present approach is effective, yielding prediction accuracy of over 80% with respect to experimental data. Subsequently, we applied such a method for prediction of T-cell epitopes in SARS coronavirus (SARS-CoV) S, N and M proteins. Based on available experimental data, the prediction accuracy is up to 90% for S protein. We suggest the use of epitope prediction methods in combination with 3D structural modelling of peptide-MHC-TCR complex to identify MHC class I restricted T-cell epitopes for use in epitope based vaccines like HIV and human cancers, which should provide a valuable step forward for the design of better vaccines and may provide in depth understanding about activation of T-cell epitopes by MHC binding peptides.     

CH3SS与OH自由基反应机理的理论研究

应用密度泛函理论(DFT)对CH3SS与OH自由基单重态反应机理进行了研究.在B3PW91/6-311+G(d,p)水平上优化了反应通道上各驻点(反应物、中间体、过渡态和产物)的几何构型,用内禀反应坐标(IRC)计算和频率分析方法对过渡态进行了验证.在QCISD(T)/6-311++G(d,p)水平上计算了各物种的单点能,并对总能量进行了零点能校正.研究结果表明,CH3SS与OH反应为多通道反应,有5条可能的反应通道.反应物首先通过不同的S—O键相互作用形成具有竞争反应机理的中间体IM1和IM2.再经过氢迁移、脱氢和裂解等机理得到主要产物P1(CH2SS+H2O),次要产物P2(CH2S+HSOH),P3(CH3SH+1SO)和P4(CH2SSO+H2),其中最低反应通道的势垒为174.6kJ.mol-1.     

Autoantibody detection by direct counting of antigen-displayed yeast cells

In this study, we report a new immunoassay platform based on yeast surface display technology for detection of autoantibodies involved in autoimmune diseases, e.g., systemic lupus erythematosus (SLE) and Sj?gren's syndrome (SS). The autoantigens of Ro52/SSA epitope and SmD were chosen to be displayed on the yeast surface with their respective antibodies as the analytes. By using magnetic beads modified with protein G, yeast cells bound with specific target antibody can be captured. The amount of analytes could be determined by counting the number of fluorescent yeast cells captured in a magnetic field. The platform showed promising results in the detection of SLE autoantibodies with high sensitivity and multiplex detection capability over the traditional approaches.     

Nuclear location of the p55 subunit of the IL-2 receptor following activation of the HT-2 T helper cell line

After mitogenic or antigenic stimulation, T cells express interleukin-2 receptors (IL-2R). The mechanism and control of signal transduction following binding of IL-2 to IL-2R are poorly understood.Using two rat monoclonal antibodies (5A2 and 7D4) specific for two distinct epitopes of the p55 subunit of mouse IL-2R, we have studied the cellular localization of this molecule by immunocytochemistry during the IL-2-mediated activation of mouse T helper cell clone HT-2. During the activation cycle, nuclear staining for the p55 subunit of the IL-2 receptor was transiently observed. It is suggested that the transient nuclear location of the IL-2R may play a critical role in the control of T-cell activation, proliferation and/or differentiation.