Synthesis, Spectral Characterization, Electrochemical, Antioxidant and Antimicrobial Evaluation of 3d-Metal Complexes of 3-Mercapto-4-(Pyren-1-ylmethylene)Amino-1,2,4-Triazin-5-One

New transition metal complexes of cobalt(II), nickel(II), copper(II) and zinc(II) with new Schiff base [HLp] derived from pyrene-1-carbaldehyde and 4-Amino-3-mercapto-5-oxo-1,2,4-triazine were synthesized and characterized by elemental analysis, 1H-NMR, FT-IR, UV-Visible, ESR spectral studies, magnetic moment measurements and cyclic voltammetry. Results prove that [HLp] acts as bidentate ligand. Low conductance data reveal non-electrolytic nature of the complexes. The spectroscopic data and magnetic moment values along with elemental analytical data support octahedral geometries for Co(II), Ni(II), Zn(II) complexes and square planar geometry for Cu(II) complexes. ESR spectra of Cu(II) complexes exhibited g values trend as g∥>g⊥> ge. The nephelauxetic ratio (β) along with orbital reduction factors (K values) report the covalent nature of the metal-ligand bonds in complexes. Thermal analysis was also carried out to propose the composition of complexes and to determine their thermal stability. Coats-Redfern method was employed to evaluate some kinetic parameters of thermal degradation processes. In vitro antimicrobial properties of all synthesized compounds were investigated. After assessing the inhibition zones, metal complexes have been found more antimicrobial active than the Schiff base against both bacterial as well as fungal strains. Metal complexes also showed remarkable fluorescence and antioxidant properties.</p

Coenzyme-B₁₂-dependent ethanolamine utilization (<i>eut</i>) genes of <i>Salmonella enterica.</i>

<p>(<b>A</b>) <i>eut</i> operon in <i>S. enterica</i>. <i>eutS</i>, <i>eutM</i>, <i>eutN</i>, <i>eutL</i> and <i>eutK</i> encode BMC shell proteins that are proposed to form the Eut microcompartment (yellow and orange) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Kofoid1" target="_blank">[20]</a>. Asterisks indicate genes that encode for enzymes with predicted N-terminal signal sequences that target them to the BMC interior <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Fan1" target="_blank">[19]</a>. Transcription is induced from the P_I promoter in the presence of both ethanolamine and vitamin B₁₂, while the promoter P_II regulates weak constitutive expression of the transcription factor EutR <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Roof1" target="_blank">[49]</a>. (<b>B</b>) Model for catabolism of ethanolamine by the Eut BMC. Ethanolamine enters the microcompartment and is metabolized to ethanol, acetyl-phosphate and acetyl-CoA, which can enter the tricarboxylic acid cycle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Kerfeld1" target="_blank">[7]</a>. Eut BMC prevents dissipation of acetaldehyde, a volatile and toxic reaction intermediate (red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342-Penrod1" target="_blank">[21]</a>. Enzymes assumed to reside in the BMC lumen include coenzyme-B₁₂-dependent ethanolamine ammonia lyase (EAL, EutBC), EAL reactivase (EutA), alcohol dehydrogenase (EutG), aldehyde dehydrogenase (EutE), and phosphotransacetylase (EutD).</p

Purification of Eut compartments.

<p>(<b>A</b>) Silver stained SDS-PAGE gel showing purification of (lane 1) Eut BMCs from <i>S. enterica</i> cells harboring EutC^1–19-EGFP, (lane 2) recombinant EutSMNLK BMCs, and (lane 3) recombinant EutS BMCs from <i>E. coli</i> C2566 cells co-expressing EutC^1–19-EGFP. Calculated protein sizes are as follows: EutS (11.6 kDa), EutM (9.8 kDa), EutN (10.4 kDa), EutL (22.7 kDa), EutK (17.5 kDa), EutC^1–19-EGFP (29.1 kDa). (<b>B</b>) Transmission electron micrographs of isolated native and recombinant Eut compartments. From left to right: Eut BMCs from <i>S. enterica</i>, EutSMNLK shells from <i>E. coli</i> C2566, EutS shells from <i>E. coli</i> C2566. (Scale bar: 100 nm).</p

Distribution of EGFP bearing putative N-terminal Eut BMC-targeting signal sequences in <i>S. enterica</i>.

<p><i>S. enterica</i> cells containing constructs for constitutive expression of EGFP, EutC^1–19-EGFP or EutG^1–19-EGFP were cultured with either glycerol or ethanolamine. Distribution of green fluorescence within the cells was observed by fluorescence microscopy. DIC images show the cell boundaries.</p

Localization of EutC^1–19-EGFP in recombinant <i>E. coli</i> expressing <i>S. enterica</i> Eut shell proteins.

<p>Fluorescence microscopy images of <i>E. coli</i> C2566 cells co-expressing EGFP or EutC^1–19-EGFP with EutS (wild type or the G39V mutant), EutMNLK or EutSMNLK. Cell boundaries are shown by the DIC images. (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342.s013" target="_blank">Table S2</a></b> for the quantification of EGFP localization in recombinant <i>E. coli</i>, and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033342#pone.0033342.s004" target="_blank">Fig. S4</a></b> for the localization of EutC^1–19-EGFP in the <i>E. coli</i> JM109 strain).</p

Hydrolysis of X-gal by <i>E. coli</i> co-expressing EutC^1–19-β-galactosidase and recombinant Eut shell proteins.

<p><i>E. coli</i> C2566 cells with constructs for constitutive expression of β-galactosidase (β-gal) or EutC^1–19-β-gal and different combinations of Eut shell proteins were grown with the β-gal substrate X-gal. Intracellular accumulation of the insoluble X-gal cleavage product was observed by Differential Interference Contrast (DIC) microscopy. Arrows point to intracellular indole deposits.</p

EutC^1–19-EGFP is sequestered in the recombinant EutSMNLK compartment.

<p>(<b>A</b>) Anti-GFP immunogold TEM of a thin section of <i>E. coli</i> JM109 cells co-expressing EutSMNLK and EutC^1–19-EGFP. Gold particles are localized to a protein shell. (Scale bar: 200 nm). (<b>B</b>) Native gel electrophoresis followed by anti-GFP western blot analysis of broken (b) and intact (i) Eut shells, harboring EutC^1–19-EGFP.</p

Formation of engineered protein shells by expression of <i>S. enterica</i> Eut shell proteins in <i>E. coli</i>.

<p>Transmission electron micrographs of thin sections of <i>S. enterica</i> and recombinant <i>E. coli</i>. (<b>A</b>) <i>S. enterica</i> grown on glycerol. (<b>B</b>) <i>S. enterica</i> grown on ethanolamine. (<b>C</b>) <i>E. coli</i> C2566 expressing recombinant EutSMNLK. (<b>D</b>) <i>E. coli</i> JM109 expressing recombinant EutSMNLK. (<b>E</b>) <i>E. coli</i> C2566 expressing recombinant EutS. (<b>F</b>) <i>E. coli</i> JM109 expressing recombinant EutS. (<b>G</b>) <i>E. coli</i> C2566 expressing recombinant EutMNLK. (<b>H</b>) <i>E. coli</i> JM109 expressing recombinant EutMNLK. Arrows indicate the location of recombinant BMCs. (Scale bar: 200 nm).</p

Additional file 1 of Bispecific T cell-engager targeting oncofetal chondroitin sulfate induces complete tumor regression and protective immune memory in mice

Additional file 1: Sup. Fig. 1. (A) ELISA showing binding of V-aCD3Mu (Coupled)(Kd = 38.8, Bmax = 3.31), rVAR2 (Kd and Bmax not determined), and V-aCD3Mu (Fused)(Kd = 14.2, Bmax = 3.36) to CSPG on a decorin backbone. Data is representative of a minimum of two separate experiments. (B) Solid 4T1 tumors 50-100 mm3 in size were treated with either PBS (n=5), V-aCD3Mu (Coupled) + CpG (n=8), or V-aCD3Mu (Fused) + CpG (n=8) on day 10, 12, 14, and 17 after tumor injection. Numbers in parentheses indicate the number of animals with complete tumor regression out of all mice in the group. Sup. Fig. 2. (A) Gating strategy on splenocytes and PBMCs in flow cytometry used to determine binding of rVAR2, aCD3Mu, V-aCD3Mu, aCD3Hu, and anti-V5 antibodies to T cells and non-T cell splenocytes/PBMCs. The gating is single cells lymphocytes live cells CD4+ and/or CD8+ cells as T cells and CD4-CD8- cells as non-T cells. The geometric MFI of the anti-penta-HIS antibodies conjugated to Alexa Flour 488 was then used to evaluate the binding of the HIS-tagged proteins. (B) Binding of aCD3Mu (Kd = 4.96, Bmax = 1.05), rVAR2 (Kd = NR, Bmax = 0.46), and V-aCD3Mu (Kd = 1.24, Bmax = 3.38) to murine recombinant CD3 in ELISA with aCD4Mu as a negative control (left). Means and standard deviations are shown. Right pane shows CSA inhibition of binding at 120 nM (right). Each dot represents one data point. Sup. Fig. 3. Cytokines measured from 4T1 and splenocyte co-culture supernatants using ELISA. Mouse splenocytes were incubated with 4T1 cancer cells together with 200 nM of the indicated protein. Sup. Fig. 4. (A) Survival curves for mice with indicated tumors treated as described in Fig. 4. The cut-off for all Kaplan-Meier plots is a tumor volume of $$\ge$$ ≥ 400 mm3. Mice were censored if they had to be excluded from the study prematurely due to reasons other than tumor size. Log-rank test was used for statistical analysis. *p < 0.05. (B) Bioluminescence in vivo imaging of C57BL/6 mice following orthotopic implantation of 5x104 Luciferase+ primary pancreatic cancer cells (CHX2000) derived from KPC mice (LSL-KrasG12D/+; p53f/f; Pdx1-Cre). Sup. Fig. 5. (A-C) Survival curves for mice treated as described in Fig. 5. The cut-off for all Kaplan-Meier plots is a tumor volume of $$\ge$$ ≥ 400 mm3. Mice were censored if they had to be excluded from the study prematurely due to reasons other than tumor size. Log-rank test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Sup. Fig. 6. (A) Treatment schedule until day 14 when spleens and tumors were harvested for flow cytometry and the subsequent gating strategy on splenocytes to evaluate different cell types in C-D. (B) Percentage of live cells relative to the PBS group in the spleen. Both CD8+ and CD4+ T cells that are CD69+, CD44hi, CD8+CD25+, or Tregs are shown. (C) UMAPs of splenocytes from all four treatment groups with clustering performed in ClusterExplorer. Cell types in clusters are explained below. Statistics were performed using one-way ANOVA with Dunnett’s post hoc test for comparison of all treatment groups to the PBS group. P values are indicated if significant or important for reading the figure. (D) Correlations between the tumor size and �8+CD69+ (p=0.67) and �4+CD69+ (p=0.14) of all live single cells in the tumor evaluated by simple linear regression. Sup. Fig. 7. Binding of mouse antibodies to 4T1 cells and B16-F10 cells in flow cytometry. Serum from C57BL/6 mice treated as described in materials and methods was diluted as illustrated on the figure and incubated with 200.000 4T1 or B16-F10 cells. Soluble CSA was added if indicated for 1 hour before detection with an anti-mouse IgG antibody conjugated to FITC

PAM1.4 does not inhibit the infected erythrocytes adhesion to CSA.

Percentage of FCR3 VAR2CSA infected erythrocytes binding to CSA in the presence of 100 μg/ml antibodies or 500 μg/ml soluble CSA (sCSA). Control, absence of antibodies; Fab, PAM1.4 Fab fragment; PAM1.4, PAM1.4 whole IgG; high and low, corresponds to total IgG purified from a pool of plasma with high and low levels of anti-VAR2CSA, respectively; IgG, IgG isotype control. Median values ± 95% CI from two independent experiments and P values using Kruskal-Wallis test followed by Dunn’s multiple comparisons test are shown. (PDF)</p