Journal: International Journal of Electrochemistry

Article Title: Electrochemical Sandwich Immunoassay for the Ultrasensitive Detection of Human MUC1 Cancer Biomarker

doi: 10.1155/2013/740265

Figure Lengend Snippet: Figure 1: Sandwich immunosensor preparation for MUC1 detection: (a) primary antibody attachment on MBs surface; (b) MBs free binding- sites blocking using BSA; (c) incubation with MUC1 antigen solutions and Ab1/MUC1 Ag complex formation on the MBs; (d) reaction with Ab2, (e) incubation with Ab3 labeled with HRP; (f) DPV measurements of MBs-bound MUC1 in presence of acetaminophen/H2O2 [19].

Article Snippet: MUC1 monoclonal mouse antibody (Ab1), MUC1 recombinant protein (Ag), and MUC1 polyclonal rabbit antibody (Ab2) were provided by Novus Biological.

Techniques: Binding Assay, Blocking Assay, Incubation, Labeling

Journal: Vaccines

Article Title: A Novel Therapeutic Tumor Vaccine Targeting MUC1 in Combination with PD-L1 Elicits Specific Anti-Tumor Immunity in Mice

doi: 10.3390/vaccines10071092

Figure Lengend Snippet: Expression and purification of recombinant protein MUC1-PDL1-IgG1 Fc (MUC1-Vax). ( A ) Plasmid-transfected E . coli lysed protein fractions were run on a 12% SDS-PAGE gel and stained with Coomassie Brilliant Blue R250. Lane M is the prestained protein molecular weight marker, lane 1 is the bacterial protein before induction, lane 2 is the bacterial protein after isopropyl β-D-1-thiogalactoside (IPTG) induction, and lane 3 is the purified MUC1-Vax protein, and lane 4 is the MUC1-Vax protein after dialysis. Recombinant protein MUC1-Vax (arrow) is indicated. ( B ) Western blot analysis of purified recombinant protein MUC1-Vax with anti-human His primary antibody. ( C ) Western blot analysis of purified recombinant protein MUC1-Vax with anti-human MUC1 antibody.

Article Snippet: These tumor cells stably expressing human MUC1 and PD-L1 were treated with 5 μg/mL or 2.5 μg/mL puromycin (Solarbio, Beijing, China) in culture medium.

Techniques: Expressing, Purification, Recombinant, Plasmid Preparation, Transfection, SDS Page, Staining, Molecular Weight, Marker, Western Blot

Journal: Vaccines

Article Title: A Novel Therapeutic Tumor Vaccine Targeting MUC1 in Combination with PD-L1 Elicits Specific Anti-Tumor Immunity in Mice

doi: 10.3390/vaccines10071092

Figure Lengend Snippet: ( A ) C57BL/6 mice were subcutaneously inoculated with 2 × 10 5 luciferase expressing LLC (LLC-MUC1-PDL1-Luc) or 3 × 10 6 Panc02 (Panc02-MUC1-PDL1-Luc) cells by hypodermic injection (I.H.). The footpads were injected with 2 × 10 6 BMDCs loaded with different proteins on the 7th and 14th days after tumor cell injection, respectively. Splenocytes of immunized mice were isolated 4 days after immunization and the activation of CD4 + T cells and CD8 + T cells (3 per group) and cytokine secretion were detected by flow cytometry. The tumor size, growth, and survival time of the remaining mice were observed (4–5 in each group). ( B ) Activation of CD4 + T cells, data are expressed as mean ± SD. ( C ) Secretion of IL-2 by CD4 + T cells, data are expressed as mean ± SD. ( D ) Secretion of IFN-γ by CD4 + T cells, data are presented as mean ± SD. ( E ) Secretion of IFN-γ by CD8 + T cells, data are presented as mean ± SD. * p < 0.05.

Article Snippet: These tumor cells stably expressing human MUC1 and PD-L1 were treated with 5 μg/mL or 2.5 μg/mL puromycin (Solarbio, Beijing, China) in culture medium.

Techniques: Luciferase, Expressing, Injection, Isolation, Activation Assay, Flow Cytometry

Journal: Vaccines

Article Title: A Novel Therapeutic Tumor Vaccine Targeting MUC1 in Combination with PD-L1 Elicits Specific Anti-Tumor Immunity in Mice

doi: 10.3390/vaccines10071092

Figure Lengend Snippet: Cell bioluminescence monitoring was performed in vivo on tumor mice inoculated with LLC-MUC1-PDL1-Luc every 6 days, as shown in figure ( A ) Bioluminescence in vivo of mice inoculated with LLC ( n = 5), blank space means the mouse died; ( B ) In vivo bioluminescence of mice inoculated Panc02 ( n = 3).

Article Snippet: These tumor cells stably expressing human MUC1 and PD-L1 were treated with 5 μg/mL or 2.5 μg/mL puromycin (Solarbio, Beijing, China) in culture medium.

Techniques: In Vivo

Journal: Plant biotechnology journal

Article Title: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice.

doi: 10.1111/j.1467-7652.2011.00614.x

Figure Lengend Snippet: Figure 1 magnICONTM viral vector transient expression system. (a) Dia- grammatic representation of magnICONTM pro-vector modules used in this study. Hsp81.1 represents the Arabidopsis heat shock protein 81.1 promoter; Integrase, Streptomyces phage PhiC31 integrase; AttP and AttB, PhiC31 integrase recombination sites; Act2, Arabidopsis actin 2 promoter; RdRP, RNA-dependent RNA polymerase; MP, movement pro- tein; LTB, synthetic LTB gene; LTB-G-MUC1, LTB gene with the GPGP linker and the MUC1 tandem repeat; LTB-G-MUC1-6His, LTB-G-MUC1 gene with a histidine tag on the C-terminus of MUC1; GFP, green fluo- rescent protein; LTBHis, LTB gene with the SGPS linker and a histidine tag, 3¢NTR, 3¢ untranslated region; NosT, nopaline synthase terminator, Int, intron; LB and RB, left and right borders of the T-DNA region. (b) Northern analysis of Nicotiana benthamiana infiltrated with magnI- CONTM pro-vector modules, for the detection of LTB-MUC1 mRNA. Lanes: 1–2, N. benthamiana infiltrated with pICH-LM1; 3, N. benthami- ana infiltrated with pICH-LTB; W.T., noninfiltrated N. benthamiana control. Bottom panel is the methylene blue-stained membrane for loading control: 10 lg of total RNA was loaded for each sample.

Article Snippet: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice Julia Pinkhasov1, M. Lucrecia Alvarez1, M. Manuela Rigano1, Khanrat Piensook1, Dalia Larios1, Martin Pabst2, Josephine Grass2, Pinku Mukherjee3, Sandra J. Gendler3, Amanda M. Walmsley1 and Hugh S. Mason1,* 1The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ, USA 2Department of Chemistry, Universitaet fuer Bodenkultur, Wien, Austria 3Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Scottsdale, AZ, USA Received 16 November 2009; revised 27 January 2011; accepted 17 February 2011.

Techniques: Plasmid Preparation, Expressing, Northern Blot, Control, Staining, Membrane

Journal: Plant biotechnology journal

Article Title: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice.

doi: 10.1111/j.1467-7652.2011.00614.x

Figure Lengend Snippet: Figure 2 Time course of LTB-MUC1 expression in magnICONTM infil- trated Nicotiana benthamiana. (a) Expression of GFP and LTB-MUC1 in N. benthamiana 7, 9 and 11 days postinfiltration (dpi). Slight necrosis is apparent in LTB-MUC1-expressing leaves 9 dpi, with complete necrosis at infiltration site by day 11. Ganglioside capture ELISA was used to quantify LTB (b) and MUC1 (c) expression at 7, 9 and 11 days postinfil- tration. Expression levels reported in micrograms per gram fresh weight (lg ⁄ g f.w.). Data are means of three independent leaf infiltrations ±SD.

Article Snippet: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice Julia Pinkhasov1, M. Lucrecia Alvarez1, M. Manuela Rigano1, Khanrat Piensook1, Dalia Larios1, Martin Pabst2, Josephine Grass2, Pinku Mukherjee3, Sandra J. Gendler3, Amanda M. Walmsley1 and Hugh S. Mason1,* 1The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ, USA 2Department of Chemistry, Universitaet fuer Bodenkultur, Wien, Austria 3Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Scottsdale, AZ, USA Received 16 November 2009; revised 27 January 2011; accepted 17 February 2011.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Plant biotechnology journal

Article Title: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice.

doi: 10.1111/j.1467-7652.2011.00614.x

Figure Lengend Snippet: Figure 3 Western blot analysis of metal affinity-purified magnICONTM-expressed LTB-6His and LTB-MUC1-6His. (a) Anti-LTB antibody was used to detect LTB-6His (lane LTB) and LTB-MUC1-6His (lane LM1). The LTB lane showed the LTB monomer at the expected molecular mass of 12 kDa as well as a possible dimer at 24 kDa. The LM1 lane showed the expected size of the LTB-MUC1-6His monomer at 16 kDa, as well as two higher molecular mass proteins at approximately 18 and 20 kDa. The protein detected at approximately 32 kDa may represent the dimer for the LTB-MUC1-6His. (b) Nickel-conjugated HRP was used to detect LTB-6His (lane LTB) and LTB-MUC1-6His (lane LM1). The LTB lane showed the His-tagged LTB monomer at 12 kDa. The LM1 lanes only showed the higher molecular mass LTB-MUC1-6His proteins, approximately 18 and 20 kDa, but not the 16-kDa protein. (c) Anti-MUC1 BC2 monoclonal antibody was used to detect LTB-6His (lane LTB) and LTB-MUC1-6His (lane LM1). No MUC1 protein was detected in the LTB lane. Only the approximately 18 and 20 kDa of LTB-MUC1-6His were detected by the MUC1-specific antibody.

Article Snippet: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice Julia Pinkhasov1, M. Lucrecia Alvarez1, M. Manuela Rigano1, Khanrat Piensook1, Dalia Larios1, Martin Pabst2, Josephine Grass2, Pinku Mukherjee3, Sandra J. Gendler3, Amanda M. Walmsley1 and Hugh S. Mason1,* 1The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ, USA 2Department of Chemistry, Universitaet fuer Bodenkultur, Wien, Austria 3Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Scottsdale, AZ, USA Received 16 November 2009; revised 27 January 2011; accepted 17 February 2011.

Techniques: Western Blot

Journal: Plant biotechnology journal

Article Title: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice.

doi: 10.1111/j.1467-7652.2011.00614.x

Figure Lengend Snippet: Figure 4 Glycosylation analysis of LTB-Muc1-6His. (a) Coomassie-stained SDS–PAGE gel of purified LTB-6His (lanes 1 and 2) and LTB-MUC1-6His (lanes 4 and 5) protein fractions. Gel bands at approximately 12 kDa (LTB-6His) and 16, 18 and 20 ⁄ 22 kDa (LTB-Muc1-6His) were excised and hydrolysed with TFA to release monosaccharides. (b) RP-HPLC of LTB-MUC1-6His shows a distinct peak for arabinose from the 20 ⁄ 22 and 18-kDa molecular weight fractions, and a shorter peak from the 16-kDa fraction. An excised piece from the gel served as a negative control (Gel blank). (c) RP-HPLC of LTB-6His does not show any monosaccharide peaks, suggesting the LTB is not glycosylated. The standard contained the following glycans: GlcN, Gluco- samine; ManN, Mannosamine; GalN, Galactosamine; Gal, Galactose; Man, Mannose; Glc, Glucose; Ara, Arabinose; Xyl, Xylose. Gel blank was used as a negative control.

Article Snippet: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice Julia Pinkhasov1, M. Lucrecia Alvarez1, M. Manuela Rigano1, Khanrat Piensook1, Dalia Larios1, Martin Pabst2, Josephine Grass2, Pinku Mukherjee3, Sandra J. Gendler3, Amanda M. Walmsley1 and Hugh S. Mason1,* 1The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ, USA 2Department of Chemistry, Universitaet fuer Bodenkultur, Wien, Austria 3Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Scottsdale, AZ, USA Received 16 November 2009; revised 27 January 2011; accepted 17 February 2011.

Techniques: Glycoproteomics, Staining, SDS Page, Molecular Weight, Negative Control

Journal: Plant biotechnology journal

Article Title: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice.

doi: 10.1111/j.1467-7652.2011.00614.x

Figure Lengend Snippet: Figure 5 Pronase peptide analysis of LTB-MUC1-6His by MS and MS ⁄ MS. (a) Amino acid sequence of LTB-MUC1-6His. Numbers at left indicate the amino acid positions from the N-terminus. The amino acid sequence GSTAPPAHG that is glycosylated with arabinose is bold ⁄ italic ⁄ dotted underlined; the tri-arabinan is found on the double proline site within this sequence. Other prolines are bold ⁄ underlined, and double prolines are bold ⁄ double underlined. (b) MS and MS ⁄ MS analysis of pronase digestion products. LTB-MUC1-6His gel fractions were pooled and subjected to digestion with pronase. One small peptide with the amino acid sequence GSTAPPAHG comprising proline residues 113 and 114 was identified with three arabinose residues attached. This glycopeptide was only found in LTB-MUC1-6His samples but not in LTB-6His.

Article Snippet: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice Julia Pinkhasov1, M. Lucrecia Alvarez1, M. Manuela Rigano1, Khanrat Piensook1, Dalia Larios1, Martin Pabst2, Josephine Grass2, Pinku Mukherjee3, Sandra J. Gendler3, Amanda M. Walmsley1 and Hugh S. Mason1,* 1The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ, USA 2Department of Chemistry, Universitaet fuer Bodenkultur, Wien, Austria 3Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Scottsdale, AZ, USA Received 16 November 2009; revised 27 January 2011; accepted 17 February 2011.

Techniques: Sequencing, Glycoproteomics

Journal: Plant biotechnology journal

Article Title: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice.

doi: 10.1111/j.1467-7652.2011.00614.x

Figure Lengend Snippet: Figure 6 Analysis of Ara-Hyp species in LT-MUC1-6His. (a) Carbon- LC-ESI-MS analysis of LTB-MUC1-6His after alkaline degradation. A high percentage of the prolines as hydroxyprolines (33%) or glycosylated hydroxyprolines, mainly with a tri-arabinan (11% of total prolines) were observed. Hyp and glyco-Hyp give two peaks containing different con- formers. (b) Quantitative comparison of the content of Proline (Pro), hydroxyproline (Hyp) and glycosylated hydroxyproline between LTB-6His and LTB-MUC1-6His. Both samples contained proline and hydroxypro- line. LTB-MUC1-6His contained more hydroxyproline than LTB-6His and contained Ara-Hyp glycopeptides, mainly with three arabinose residues. Ara2, Ara3 and Ara4 designate a Hyp with 2, 3 and 4 Ara residues, respectively.

Article Snippet: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice Julia Pinkhasov1, M. Lucrecia Alvarez1, M. Manuela Rigano1, Khanrat Piensook1, Dalia Larios1, Martin Pabst2, Josephine Grass2, Pinku Mukherjee3, Sandra J. Gendler3, Amanda M. Walmsley1 and Hugh S. Mason1,* 1The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ, USA 2Department of Chemistry, Universitaet fuer Bodenkultur, Wien, Austria 3Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Scottsdale, AZ, USA Received 16 November 2009; revised 27 January 2011; accepted 17 February 2011.

Techniques: Comparison

Journal: Plant biotechnology journal

Article Title: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice.

doi: 10.1111/j.1467-7652.2011.00614.x

Figure Lengend Snippet: Figure 7 Parenteral immunization of MUC1.Tg mice with purified magnICONTM generated LTB- MUC1-6His. (a) Serum analysis for MUC1-specific IgG in MUC1.Tg mice treated with: phosphate buffer saline vehicle control (PBS); 100 lg CpG ODN (CpG); 100 lg CpG + LTB-MUC1-6His (LM1CpG). Serum samples were derived from blood drawn 1 week after each immunization (days 0, 7 and 14) and boost (day 35). (b) Serum MUC1-specific IgG1 and IgG2a (c) in MUC1.Tg mice treated with: PBS; 100 lg CpG ODN (CpG); LTB-MUC1-6His (LTB-MUC1); 100 lg CpG + LTB- MUC1-6His (CpG + LTB-MUC1). Anti-MUC1 titres (IgG, IgG1 and IgG2a) in mice treated with CpG + LTB-MUC1 are significantly higher (P < 0.05) than the PBS control group. Individual mouse data are shown (n = 3–5 mice per group).

Article Snippet: Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice Julia Pinkhasov1, M. Lucrecia Alvarez1, M. Manuela Rigano1, Khanrat Piensook1, Dalia Larios1, Martin Pabst2, Josephine Grass2, Pinku Mukherjee3, Sandra J. Gendler3, Amanda M. Walmsley1 and Hugh S. Mason1,* 1The Biodesign Institute and School of Life Sciences, Arizona State University, Tempe, AZ, USA 2Department of Chemistry, Universitaet fuer Bodenkultur, Wien, Austria 3Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Mayo Clinic, Scottsdale, AZ, USA Received 16 November 2009; revised 27 January 2011; accepted 17 February 2011.

Techniques: Generated, Saline, Control, Derivative Assay