Time for Singapore to Relook Abortion Law

Figure S1. Effect of anti-ITGA2 antibody on cell morphology. The AGS cells were treated with a 3 Οg of the anti-ITGA2 antibodies or isotype control antibodies (negative control) for 48 h, and cell morphology was observed at 200X magnification. Data are representative of three independent experiments. (PPTX 1463 kb

Solubility and secretion of αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR in different components.

<p>The transformed cells were induced with IPTG and the presence of αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR were detected by western blot analysis using an anti-histidine tag antibody in (a) concentrated LB medium, (b) soluble lysate, and (c) insoluble protein (pellet), as described in the Materials and Methods section. Lane 1, BL21 as negative control; Lane 2, αEGFR-osmY/BL21; Lane 3, osmY-αEGFR/BL21; and Lane 4, pelB-αEGFR/BL21.</p

Subcellular localization of scFv fusion protein.

<p>LB: growth medium. S: soluble lysate. P: pellet.</p><p>The distribution of osmY-scFv, scFv-osmY, and pelB-scFv in the bacteria was observed by western blot analysis, and the intensity was estimated by densitometry. The protein quantity was calculated on the basis of protein concentration folds and intensity ratios and presented as a percentage.</p

Construction and expression of secreted αEGFR.

<p>(a) αEGFR was fused with the N- or C-terminus of the <i>osmY</i> gene to form αEGFR-osmY and osmY-αEGFR fusion proteins, respectively. αEGFR expressed in the periplasmic space (pelB-αEGFR) was used as the control. H, Histidine tag. (b) αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR plasmids were transformed into BL-21 to obtain αEGFR-osmY/BL21, osmY-αEGFR/BL21, and pelB-αEGFR/BL21 cells, respectively. The expression of αEGFR was confirmed by western blot analysis using an anti-histidine tag antibody. Lane 1, BL21 as negative control; Lane 2, αEGFR-osmY/BL21; Lane 3, osmY-αEGFR/BL21; and Lane 4, pelB-αEGFR/BL21.</p

Development of a bacterial secretion system for the production of a soluble and secreted single-chain antibody.

<p>scFv was fused with the bacterial secretory carrier protein osmY to produce a good yield of soluble scFv secreted into the LB medium and to circumvent scFv inclusion body formation in the cytoplasm.</p

Antigen-binding activity of αEGFR-osmY, osmY-αEGFR, and refolded pelB-αEGFR.

<p>The fusion protein αEGFR-osmY and osmY-αEGFR were purified by using a Ni-column, and the control pelB-αEGFR was <b><i>purified</i></b> by using the Ni-column under denaturing/refolding conditions. The binding activities of different concentrations of αEGFR against EGFR-positive cells (MDA-MB-468) were determined by ELISA using an anti-histidine tag antibody.</p

Function of secreted αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR.

<p>The growth medium of αEGFR-osmY/BL21, osmY-αEGFR/BL21, and pelB-αEGFR/BL21 was added to the EGFR-positive MDA-MB-468 cells, and the binding activity of the αEGFR fusion protein was detected by ELISA using an anti-histidine tag antibody.</p

An Activity-Based Near-Infrared Glucuronide Trapping Probe for Imaging β-Glucuronidase Expression in Deep Tissues

β-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of β-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of β-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol–glucuronide moiety (TrapG) to trap the fluorochromes in the vicinity of the active enzyme. β-glucuronidase-mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the fluorochrome to nucleophilic moieties located near β-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified β-glucuronidase or β-glucuronidase-expressing CT26 cells (CT26/mβG) but not on bovine serum albumin or non-β-glucuronidase-expressing CT26 cells used as controls. β-glucuronidase-activated FITC-TrapG did not interfere with β-glucuronidase activity and could label bystander proteins near β-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/mβG tumors, but only NIR-TrapG could image CT26/mβG tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing β-glucuronidase activity in vivo

Additional file 4: of Blockade of ITGA2 Induces Apoptosis and Inhibits Cell Migration in Gastric Cancer

Figure S2. Low dose of anti-ITGA2 antibodies did not induce cell death in AGS cells. Photography and quantitative analyses on cell number of the AGS cells treated with 0.1 μg anti-ITGA2 antibodies or isotype control antibodies (negative control) for 18 h. Data are expressed as mean ± standard deviation (S.D). Statistical comparisons were made by one-way ANOVA with Bonferroni comparisons. Data are representative of three independent experiments. (PPTX 784 kb

Development of a Gd(III)-Based Receptor-Induced Magnetization Enhancement (RIME) Contrast Agent for β‑Glucuronidase Activity Profiling

β-Glucuronidase is a key lysosomal enzyme and is often overexpressed in necrotic tumor masses. We report here the synthesis of a pro receptor-induced magnetization enhancement (pro-RIME) magnetic resonance imaging (MRI) contrast agent ([Gd­(DOTA-FPβGu)]) for molecular imaging of β-glucuronidase activity in tumor tissues. The contrast agent consists of two parts, a gadolinium complex and a β-glucuronidase substrate (β-d-glucopyranuronic acid). The binding association constant (<i>K</i>_A) of [Gd­(DOTA-FPβGu)] is 7.42 × 10², which is significantly lower than that of a commercially available MS-325 (<i>K</i>_A = 3.0 × 10⁴) RIME contrast agent. The low <i>K</i>_A value of [Gd­(DOTA-FPβGu)] is due to the pendant β-d-glucopyranuronic acid moiety. Therefore, [Gd­(DOTA-FPβGu)] can be used for detection of β-glucuronidase through RIME modulation. The detail mechanism of enzymatic activation of [Gd­(DOTA-FPβGu)] was elucidated by LC-MS. The kinetics of β-glucuronidase catalyzed hydrolysis of [Eu­(DOTA-FPβGu)] at pH 7.4 best fit the Miechalis–Menten kinetic mode with <i>K</i>_m = 1.38 mM, <i>k</i>_cat = 3.76 × 10³, and <i>k</i>_cat/<i>K</i>_m = 2.72 × 10³ M^–1 s^–1. The low <i>K</i>_m value indicates high affinity of β-glucuronidase for [Gd­(DOTA-FPβGu)] at physiological pH. Relaxometric studies revealed that <i>T</i>₁ relaxivity of [Gd­(DOTA-FPβGu)] changes in response to the concentration of β-glucuronidase. Consistent with the relaxometric studies, [Gd­(DOTA-FPβGu)] showed significant change in MR image signal in the presence of β-glucuronidase and HSA. <i>In vitro</i> and <i>in vivo</i> MR images demonstrated appreciable differences in signal enhancement in the cell lines and tumor xenografts in accordance to their expression levels of β-glucuronidase