Determination of the U937 cell viability.

<p>The cell viability was determined 30 min after the addition of 5 mM H₂O₂ or Fenton reagent to the U937 cells. The results are normalized to control U937 cells. The data are presented as the mean and standard deviation of at least 3 measurements.</p

Detection of singlet oxygen by EPR spin-trapping spectroscopy.

<p>TEMPONE EPR spectra were measured in the control (A and B), the H₂O₂-treated (C and D) and the Fenton reagent-treated (E and F) U937 cells in the presence of 100 mM TEMPD. U937 cells were treated with no addition (A), 5 mM H₂O₂ (C) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (E) for a period indicated in Fig. In C and E, top traces show the simulation of TEMPONE EPR signal using hyperfine coupling constants <i>a</i>^N = 16 G. Chemical source of ¹O₂ (10 mM molybdic acid + 10 mM H₂O₂ measured right after the preparation) was used as a positive control (C,E). Bar graphs represent the hight of the middle peak of TEMPONE EPR signal in the control (B), the H₂O₂-treated (D) and the Fenton reagent-treated (F) U937 cells. Experimental EPR conditions were as follows: microwave power, 10 mW; modulation amplitude, 1 G; modulation frequency, 100 kHz; sweep width, 100 G; scan rate, 1.62 G s-1, gain 500. Bars represent 4000 (A) and 8000 (C and E) relative units. Data are presented as mean values and standard deviations. The mean value represent the average value from at least three measurements.</p

Analysis of DNA strand breaks by Comet assay.

<p>Comet assay of the control (A), the H₂O₂-treated (B) and the Fenton reagent-treated (C) U937 cells. The U937 cells were treated with 5 mM H₂O₂ (B) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (C) for 30 min. After the treatment, U937 cells were stained by SYBR Green.</p

Analysis of the medians of comet, head, and tail parameters in the control, the H₂O₂-treated and the Fenton reagent-treated U937 cells.

<p>Data are presented as mean values and standard deviations. The mean value represents the average value from at least three measurements.</p><p>Analysis of the medians of comet, head, and tail parameters in the control, the H₂O₂-treated and the Fenton reagent-treated U937 cells.</p

Detection of lipid peroxidation product malondialdehyde by HPLC analysis.

<p>The chromatogram of DNPH-MDA complex in U937 cells (A) and DNPH-MDA standard (B). In A, chromatogram of DNPH-MDA complex was measured in the control (trace a), the H₂O₂-treated (trace b) and the Fenton reagent-treated (trace c) U937 cells. The U937 cells were treated with 5 mM H₂O₂ (b) and Fenton reagent (5 mM H₂O₂ and 1 mM FeSO₄) (c) for 30 min. After the treatment, lipids were separated from proteins and DNPH was added to lipids. In B, the chromatogram of DNPH-MDA standard shows the retention time of 3 min 50 s. The insert shows the dependence of average peak area on the concentration of DNPH-MDA standard. Based on the calibration curve, the concentrations of DNPH-MDA complex determined from calibration curve were as following: 0.029±0.003 nmol ml⁻¹ (control), 0.030±0.003 (H₂O₂) and 0.09±0.02 nmol ml⁻¹ (Fenton reagent). The coefficient of determination R² was determined as 0.9997. Data are presented as mean values and standard deviations. The mean value represents the average value from at least three measurements.</p

DataSheet_1_Myomedin replicas of gp120 V3 loop glycan epitopes recognized by PGT121 and PGT126 antibodies as non-cognate antigens for stimulation of HIV-1 broadly neutralizing antibodies.docx

IntroductionImprinting broadly neutralizing antibody (bNAb) paratopes by shape complementary protein mimotopes represents a potential alternative for developing vaccine immunogens. This approach, designated as a Non-Cognate Ligand Strategy (NCLS), has recently been used for the identification of protein variants mimicking CD4 binding region epitope or membrane proximal external region (MPER) epitope of HIV-1 envelope (Env) glycoprotein. However, the potential of small binding proteins to mimic viral glycan-containing epitopes has not yet been verified.MethodsIn this work, we employed a highly complex combinatorial Myomedin scaffold library to identify variants recognizing paratopes of super candidate bNAbs, PGT121 and PGT126, specific for HIV-1 V3 loop epitopes.ResultsIn the collection of Myomedins called MLD variants targeted to PGT121, three candidates competed with gp120 for binding to this bNAb in ELISA, thus suggesting an overlapping binding site and epitope-mimicking potential. Myomedins targeted to PGT126 designated MLB also provided variants that competed with gp120. Immunization of mice with MLB or MLD binders resulted in the production of anti-gp120 and -Env serum antibodies. Mouse hyper-immune sera elicited with MLB036, MLB041, MLB049, and MLD108 moderately neutralized 8-to-10 of 22 tested HIV-1-pseudotyped viruses of A, B, and C clades in vitro.DiscussionOur data demonstrate that Myomedin-derived variants can mimic particular V3 glycan epitopes of prominent anti-HIV-1 bNAbs, ascertain the potential of particular glycans controlling neutralizing sensitivity of individual HIV-1 pseudoviruses, and represent promising prophylactic candidates for HIV-1 vaccine development.</p