Journal: Military Medical Research

Article Title: Octopus-inspired engineered bacteria with a plug-and-play surface display system achieves enhanced tumor-specific colonization and antitumor immunity

doi: 10.1016/j.mmr.2026.100030

Figure Lengend Snippet: Schematic of the construction and therapeutic mechanism of the octopus-inspired triple-engineered bacteria (OITE strain) . The antitumor attenuated Salmonella typhimurium AISI strain was triple-engineered. (1) ST/SC-RGD×4 “tentacles”-Surface modification: the ST sequence was expressed within the external third loop of OmpA in the AISI strain, resulting in the AISI-ST strain. The ST protein was coexpressed with OmpA (OmpA-ST fusion protein) localized to the bacterial outer membrane. An incubation with SC-RGD×4 protein (SC-RGD×4) led to the formation of AISI-ST/SC-RGD×4 strain (AISI-ST/SC-RGD×4) through ST-SC-mediated covalent conjugation. (2) Dynamic EPS “camouflage”-Immunoactivation engineering: the AISI-H-ST strain was created by introducing the quorum-sensing (QS) promoter pLuxI to control HtrA expression into AISI-ST strain, which specifically increased HtrA-mediated extracellular polysaccharide (EPS) production to amplify bacteria-mediated immune activation. (3) Anti-PD1nanobody (PD1nb) “secretion”-Checkpoint blockade engineering: further programming of AISI-H-ST strain enabled QS-triggered anti-PD1nb secretion, generating the AISI-HP-ST strain. The incubation of AISI-HP-ST strain with SC-RGD×4 protein finally produced the OITE strain (AISI-HP-ST/SC-RGD×4). The intravenously administered OITE strain highly selectively accumulates in tumors through RGD-αvβ3 integrin interactions, with subsequent bacterial proliferation initiating two therapeutic actions: HtrA-mediated immune activation via increasing immune cells infiltration and activation, and secreted PD1nb-based blockade of PD-1/PD-L1 immunosuppressive signaling. OITE. Octopus-inspired, triple-engineered bacterium; AISI. Attenuated Salmonella Δ htrA :: luxI - VNP20009 strains; ST. SpyTag; SC. SpyCatcherΔ; RGD. Arginine-glycine-aspartic acid; HtrA. High-temperature requirement A; PD1. Programmed cell death protein 1; H. HtrA; HP. HtrA and PD1nb; AHL. N-acyl homoserine lactone; MACS. Macrophages; Teffs. Effector T cells; Tregs. Regulatory T cells.

Article Snippet: To prepare the surfaces for the strain adhesion assays, the recombinant mouse integrin αvβ3 (TGAV & ITGB3) heterodimer protein (HY-P700761, MCE, USA) was first diluted to 10 μg/ml in 50 mmol/L carbonate buffer (pH 9.6).

Techniques: Bacteria, Modification, Sequencing, Membrane, Incubation, Conjugation Assay, Control, Expressing, Activation Assay, Produced

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Self-Triggered Apoptosis Enzyme Prodrug Therapy (STAEPT): Enhancing Targeted Therapies via Recurrent Bystander Killing Effect by Exploiting Caspase-Cleavable Linker.

doi: 10.1002/advs.201800368

Figure Lengend Snippet: Figure 2. Enhanced cellular uptake and cytotoxic activity of RGDEVD-DOX in integrin αvβ3 expressing cells. a) Representative confocal images (left) and quantitative analysis (right) of HDMEC and U-87 MG cells exposed to fluorescent-labeled RDEVD and RGDEVD peptides. b) Representative confocal images (left) and quantitative analysis (right) of scrambled control and integrin αv (ITGAV) siRNA-transfected HDMECs and U-87 MG cells exposed to fluorescent-labeled RGDEVD peptide. Green and blue indicate the fluorescent-labeled peptides and cell nuclei, respectively. Scale bar, 50 µm. c) Flow cytometry analysis of isotype control (top), scrambled control-transfected (lower left), and ITGAV siRNA-transfected (lower right) U87 MG cells incubated with fluorescent-labeled RGDEVD and stained with an antibody against integrin αvβ3. d) Representative confocal images (left) and quantitative analysis (right) of U-87 MG and HT-29 cells treated with RDEVD-DOX and RGDEVD-DOX. Red and blue indicate the intrinsic red fluorescence of doxorubicin and cell nuclei, respectively. Scale bar, 50 µm. e) Concentration-dependent cytotoxicity of RDEVD-DOX and RGDEVD-DOX on U-87 MG (left) and HT-29 (right) determined by MTT assay (n = 4). f) Doxorubicin release from RGDEVD-DOX when incubated in PBS (pH 7.4) containing (or not containing) carboxylesterase (n = 3). Data are mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The cells were incubated with Alexa Fluor 488-conjugated integrin αvβ3 antibody (1:100; R&D Systems, Minneapolis, MN; Cat. No. FAB3050G) for an hour at 4 °C, washed, and suspended in PBS containing 0.5% BSA.

Techniques: Activity Assay, Expressing, Labeling, Control, Transfection, Flow Cytometry, Incubation, Staining, Fluorescence, Concentration Assay, MTT Assay

Journal: International Journal of Nanomedicine

Article Title: Analysis of in situ and ex vivo αVß3 integrin expression during experimental carotid atherogenesis

doi: 10.2147/ijn.s28065

Figure Lengend Snippet: Figure 4 Accumulation of macrophages in the stenotic segment. (A) Representative micrograph of carotid artery sections stained with antibodies against αvβ3 integrin (400× magnification, scale bar = 100 µm). (B) Representative images of MAC-3 immunostaining in the stenotic lesion areas of the left carotid artery (400× magnification, scale bar = 100 µm). (C) Quantitative analyses of MAC-3-positive cells. (D) Colocalization in the proximal neointima, blue fluorescence of DAPI, the red fluorescence of Cy5.5 (probe) and the green fluorescence of FITC (macrophage) were examined (800× magnification). Notes: Upper panel shows representative photomicrographs of cross sections of left carotid artery proximal carotid site, white arrows show positive staining. Lower panel shows representative photomicrographs of cross sections of right carotid artery. Abbreviation: DAPI, diamidino-2-phenylindole dihydrochloride.

Article Snippet: Primary antibody against α V β 3 integrin (anti-human integrin α V β 3 CD51/CD61, R&D Systems; Minneapolis, MN, 1:200) was used for immunostaining of α V β 3 integrin.

Techniques: Staining, Immunostaining, Fluorescence

Journal: Biochemistry

Article Title: Identification of Immunoreactive Regions of Homology between Soluble Epidermal Growth Factor Receptor (sEGFR) and ?5-Integrin †

doi: 10.1021/bi200126j

Figure Lengend Snippet: Summary of amino acid sequences as determined by LC ESI MS/MS using fractions enriched for p140, as described in the text.

Article Snippet: Recombinant α5/β1 integrin and αv/β3 integrin were purchased from R&D Systems, Inc (Minneapolis, MN).

Techniques:

Journal: Biochemistry

Article Title: Identification of Immunoreactive Regions of Homology between Soluble Epidermal Growth Factor Receptor (sEGFR) and ?5-Integrin †

doi: 10.1021/bi200126j

Figure Lengend Snippet: Expression of BASP-1, α5-integrin, and β1-intergin in JEG-3 and HEY cell lysates as detected by immunoblot analysis. JEG-3 (panel A) and HEY (panel B) cell lysates were resolved by SDS-PAGE as a single band spanning the width of the gel, and transferred to PVDF membrane. The membrane was immunoblotted using a multichannel Immentics blotter, with antibodies directed against BASP-1, α5-integrin, β1-integrin, sEGFR, and EGFR (cytoplasmic domain) at the indicated dilutions. Solid arrowheads indicate migration of BASP-1, α5-integrin, β1-integrin, and EGFR, respectively. Open arrowheads indicate migration of p140. Of the tested species, only a band reactive with anti-α5-integrin co-migrated with p140.

Article Snippet: Recombinant α5/β1 integrin and αv/β3 integrin were purchased from R&D Systems, Inc (Minneapolis, MN).

Techniques: Expressing, Western Blot, SDS Page, Migration

Journal: Biochemistry

Article Title: Identification of Immunoreactive Regions of Homology between Soluble Epidermal Growth Factor Receptor (sEGFR) and ?5-Integrin †

doi: 10.1021/bi200126j

Figure Lengend Snippet: Anti-sEGFR recognizes α5-integrin. Panel A. Immunoblot of p140 immunoprecipitated from JEG-3 cell lysates with anti-α5-integrin antibody and reacted with anti-sEGFR (right panel) or secondary antibody alone (left panel). Solid arrowhead indicates mobility of p140. Open arrowhead indicates mobility of a minor ~95 kDa band, which we postulate is a product of α5-integrin. Panel B. Immunoblot of recombinant α5/β1-integrin reacted with different dilutions of anti-sEGFR, as indicated, confirming that anti-sEGFR recognizes α5-integrin.

Article Snippet: Recombinant α5/β1 integrin and αv/β3 integrin were purchased from R&D Systems, Inc (Minneapolis, MN).

Techniques: Western Blot, Immunoprecipitation, Recombinant

Journal: Biochemistry

Article Title: Identification of Immunoreactive Regions of Homology between Soluble Epidermal Growth Factor Receptor (sEGFR) and ?5-Integrin †

doi: 10.1021/bi200126j

Figure Lengend Snippet: Expression of α5-integrin is detected by immunoblot analysis with anti-sEGFR and anti-α5-integrin antibodies. α5-integrin deficient CHO B2 cells express very low endogenous α5-integrin as detected by anti-sEGFR (Panel A) or anti-α5-integrin (Panel B). Lysates of CHO B2 cells stably expressing human α5-integrin (CHO-α5) contain species reactive as a band by reducing SDS-PAGE followed by anti-sEGFR immunoblot, or a higher molecular mass band by non-reducing SDS-PAGE followed by anti-α5-integrin immunoblot (following manufacturer’s instructions for this antibody). CHO-sEGFR and JEG-3 cells are included as negative and positive controls for p140/α5-integrin, respectively. Note different exposure lengths for CHO-sEGFR and JEG-3 vs. CHO B2 and CHO-α5 lanes in Panel A for clarity.

Article Snippet: Recombinant α5/β1 integrin and αv/β3 integrin were purchased from R&D Systems, Inc (Minneapolis, MN).

Techniques: Expressing, Western Blot, Stable Transfection, SDS Page

Journal: Biochemistry

Article Title: Identification of Immunoreactive Regions of Homology between Soluble Epidermal Growth Factor Receptor (sEGFR) and ?5-Integrin †

doi: 10.1021/bi200126j

Figure Lengend Snippet: Panel A. Diagram summary of α5-integrin amino acid sequence. Predicted sites of CNBr cleavage are shown with arrowheads. Peptides recovered by LC ESI MS/MS from a anti-sEGFR 31-mer immunoreactive 27-kDa gel band generated by CNBr hydrolysis are underlined: 695VTAPPEAEYSGLVR708 and 739AGASLWGGLR748. Potential sites of N-linked glycosylation are highlighted in bold. Potential sites of cyanogen bromide cleavage are indicated with solid arrowheads. Panel B. Sequence comparison of α5-integrin residues 588–737 with the sEGFR 31-mer peptide. Regions of α5-integrin that have >35% identity with sEGFR were aligned according to SIM analysis (http://www.expasy.org/tools/sim-prot.html). Amino acids sharing sequence identity are highlighted with an asterisk.

Article Snippet: Recombinant α5/β1 integrin and αv/β3 integrin were purchased from R&D Systems, Inc (Minneapolis, MN).

Techniques: Sequencing, Tandem Mass Spectroscopy, Generated

Journal: Biochemistry

Article Title: Identification of Immunoreactive Regions of Homology between Soluble Epidermal Growth Factor Receptor (sEGFR) and ?5-Integrin †

doi: 10.1021/bi200126j

Figure Lengend Snippet: Immunoblots of recombinant α5/β1 integrin digested by CNBr hydrolysis. Panel A. Time-course of CNBr hydrolysis of recombinant α5/β1 integrin. CNBr hydrolysis was terminated at the times indicated. A precursor-product relationship is evident resulting in an anti-sEGFR-reactive band of ~27-kDa. Panel B. CNBr hydrolysis of deglycosylated α5-integrin. Prior to CNBr hydrolysis, recombinant α5/β1-integrin was treated with PNGaseF. An anti-sEGFR-reactive protein of ~17-kDa was generated by this procedure; this peptide corresponds to α5-integrin peptide 588–737 (see Figure 7), indicating that the anti-sEGFR epitope of α5-integrin is located between amino acids 588–737.

Article Snippet: Recombinant α5/β1 integrin and αv/β3 integrin were purchased from R&D Systems, Inc (Minneapolis, MN).

Techniques: Western Blot, Recombinant, Generated

Journal: Biochemistry

Article Title: Identification of Immunoreactive Regions of Homology between Soluble Epidermal Growth Factor Receptor (sEGFR) and ?5-Integrin †

doi: 10.1021/bi200126j

Figure Lengend Snippet: Surface plasmon resonance sensorgrams of anti-sEGFR binding to a sEGFR vs. two α5-integrin peptides is indicated. Absorbance in relative units (RU) is plotted vs. time (sec) for immobilized anti-sEGFR in the presence of a sEGFR 31-mer peptide 604–634 (panel A), a α5-integrin peptide 637–649 (panel B), and a α5-integrin peptide 710–735 (panel C). Graphs of RU vs. peptide concentration are included as insets.

Article Snippet: Recombinant α5/β1 integrin and αv/β3 integrin were purchased from R&D Systems, Inc (Minneapolis, MN).

Techniques: SPR Assay, Binding Assay, Concentration Assay

Journal: Biochemistry

Article Title: Identification of Immunoreactive Regions of Homology between Soluble Epidermal Growth Factor Receptor (sEGFR) and ?5-Integrin †

doi: 10.1021/bi200126j

Figure Lengend Snippet: Summary of surface plasmon resonance of sEGFR and α5-integrin peptide interactions with anti-sEGFR.

Article Snippet: Recombinant α5/β1 integrin and αv/β3 integrin were purchased from R&D Systems, Inc (Minneapolis, MN).

Techniques: SPR Assay, Sequencing, Binding Assay

Journal: Biochemistry

Article Title: Identification of Immunoreactive Regions of Homology between Soluble Epidermal Growth Factor Receptor (sEGFR) and ?5-Integrin †

doi: 10.1021/bi200126j

Figure Lengend Snippet: (A) Anti-sEGFR promotes compaction of suspended CHO B2 cells. Compaction of CHO-P3 and CHO-α5 was compared by suspending cells in hanging drops in the presence of PBS (panels A, B), rabbit IgG (panels C, D) or anti-sEGFR (panels E, F). In general, anti-sEGFR appears to markedly enhance aggregate compaction. This effect is greater for α5-integrin expressing cells than for control cell lines. (B) Quantification of aggregate size. Image analysis was used to calculate average aggregate size for parental and α5-integrin expressing cells incubated in hanging drop cultures in PBS, rabbit IgG, and anti-sEGFR. Statistical analysis by ANOVA revealed a significant difference in aggregate size in response to anti-sEGFR treatment (p<0.0001). Tukey’s Multiple Comparisons Test revealed a pair-wise difference between the following groups of cells: CHO-P3-anti-sEGFR and CHO- α5-anti-sEGFR (p<0.001); CHO-α5-IgG and CHO-α5-anti-sEGFR (p<0.01); CHO-P3-IgG and CHO-P3-anti-sEGFR (p<0.001). As expected, no difference in size was detected between CHO-P3 and CHO- P3-IgG (p>0.05) or CHO-α5 and CHO-α5 IgG (p>0.05).

Article Snippet: Recombinant α5/β1 integrin and αv/β3 integrin were purchased from R&D Systems, Inc (Minneapolis, MN).

Techniques: Expressing, Incubation