The representative images of selected peptide-fluorescent bacteria binding with tissue sections from the mice xenograft.

<p>The scale bar was 20 µm. Data were representative of at least three independent experiments.</p

The process of screening and enriching the binding peptide library for cancer cells with bacteria surface display methods.

<p>(<b>A</b>) Schematic representation of peptides screening and selection with bacterial display library. 1. Constructed the library by transforming plasmids into <i>E.coli</i> MC1061; 2. Discarded the bacteria binding with normal cells after pre-incubation; 3. Incubated the mixture of cancer cells and residual bacteria; 4. Analyzed the binding effect of cancer cells with bacteria using FACS; 5. Sorted the binding bacteria to cancer cells by flow cytometry and cultured the binding bacteria in medium; 6. Performed the next round binding bacteria screening; 7. Isolated the binding bacteria to cancer cells and sequenced the peptides displayed on the surface of the bacteria. (<b>B</b>) The progress of enrichment of bacteria binding with cancer cells by FACS in 6 screening rounds.</p

The results of selected monoclonal peptide-fluorescent bacteria specifically binding with various cells.

<p>(<b>A</b>) Fluorescence microscope images of bacteria clones incubated with cells. A549<b>^△</b> was incubated with CPX only bacteria and other cells were incubated with selected monoclonal peptide-fluorescent bacteria, the scale bar was 20 µm. (<b>B</b>) FACS results of various cells binding with monoclonal peptide-fluorescent bacteria at ratio 1∶100. (<b>C</b>) Percentage of binding fraction of monoclonal peptide-fluorescent bacteria with various cells from FACS data. Data were mean ± S.D. of at least three independent experiments.</p

Characterization of binding ability of fixed bacteria.

<p>(<b>A</b>) Percentage of binding fraction of fresh and fixed monoclonal peptide-fluorescent bacteria with various cells from FACS data. Cells binding to fresh bacteria (black) at ratio 1∶100 and binding to fixed bacteria (gray) at ratio 1∶500. (<b>B</b>) The fluorescence microscope images of A549 cells incubating with fresh bacteria and fixed bacteria at ratio 1∶500, at day 1, day 7 and day 30 after fixation and the scale bar was 20 µm. (<b>C</b>) FACS results of A549 cells binding with fresh and fixed monoclonal peptide-fluorescent bacteria at ratio 1∶500 at different time points. (<b>D</b>) Percentage of binding fraction of fresh and fixed monoclonal peptide-fluorescent bacteria with A549 cells from FACS data Cells binding with fresh bacteria and fixed bacteria at ratio 1∶500 at different time points. Data were mean ± S.D. of at least three independent experiments.</p

Sequences of peptides.

<p>Sequences and binding rates of different peptides with A549 cells. Peptides were ordered by frequency in experiments.</p

CD146 Deletion in the Nervous System Impairs Appetite, Locomotor Activity and Spatial Learning in Mice

<div><p>Cell adhesion molecules (CAMs) are crucial effectors for the development and maintenance of the nervous system. Mutations in human CAM genes are linked to brain disorders and psychological diseases, and CAM knockout mice always exhibit similar behavioral abnormalities. CD146 is a CAM of the immunoglobulin superfamily that interacts with Neurite Outgrowth Factor and involved in neurite extension <i>in vitro</i>. However, little is known about its <i>in vivo</i> function in the nervous system. In this study, we used a murine CD146 nervous system knockout (CD146^ns-ko) model. We found that the brains of some CD146^ns-ko mice were malformed with small olfactory bulbs. CD146^ns-ko mice exhibited lower body weights and smaller food intake when compared with wild type littermates. Importantly, behavior tests revealed that CD146^ns-ko mice exhibited significant decreased locomotor activity and impaired capacity for spatial learning and memory. Our results demonstrate that CD146 is important for mammalian nervous system development and proper behavior patterns.</p></div

Fenobody: A Ferritin-Displayed Nanobody with High Apparent Affinity and Half-Life Extension

Nanobodies consist of a single domain variable fragment of a camelid heavy-chain antibody. Nanobodies have potential applications in biomedical fields because of their simple production procedures and low cost. Occasionally, nanobody clones of interest exhibit low affinities for their target antigens, which, together with their short half-life limit bioanalytical or therapeutic applications. Here, we developed a novel platform we named fenobody, in which a nanobody developed against H5N1 virus is displayed on the surface of ferritin in the form of a 24mer. We constructed a fenobody by substituting the fifth helix of ferritin with the nanobody. TEM analysis showed that nanobodies were displayed on the surface of ferritin in the form of 6 × 4 bundles, and that these clustered nanobodies are flexible for antigen binding in spatial structure. Comparing fenobodies with conventional nanobodies currently used revealed that the antigen binding apparent affinity of anti-H5N1 fenobody was dramatically increased (∼360-fold). Crucially, their half-life extension in a murine model was 10-fold longer than anti-H5N1 nanobody. In addition, we found that our fenobodies are highly expressed in Escherichia coli, and are both soluble and thermo-stable nanocages that self-assemble as 24-polymers. In conclusion, our results demonstrate that fenobodies have unique advantages over currently available systems for apparent affinity enhancement and half-life extension of nanobodies. Our fenobody system presents a suitable platform for various large-scale biotechnological processes and should greatly facilitate the application of nanobody technology in these areas

Knockout of CD146 in the nervous system impaired spatial learning and memory in mice.

<p>(A) WT and CD146^ns-ko mice at the age of 1 month were subjected to the Morris water maze test. During the 7-day training period, time spent in finding the target of each mouse was recorded and analyzed with a repeated measures ANOVA. At the last day of probe test, distance swam (B) and time spent in each quadrant by the mice (C) were recorded and analyzed with Student’s t test. The brains of 1-month-old WT and CD146^ns-ko mice were sagittally sectioned. The hippocampal area (D) and neuron density of CA1, CA3 and DG (E) were measured after Nissl staining. Data is presented as means ± SEM.</p

Knockout of CD146 in the nervous system resulted in decreased body weight and food intake in mice.

<p>Body weight of male (A) and female (B) mice at the ages of 1 month, 3 months and 6 months were measured and analyzed with a two-way ANOVA. Daily food intake of male (C) and female (D) mice at the age of 1 month was measured once a week for 4 weeks. Data is presented as means ± SEM and analyzed by a repeated measures ANOVA.</p

CD146^ns-ko mice show stochastic reduction in the size of olfactory bulbs.

<p>(A) Representative images of WT brain, CD146^ns-ko normal brain and CD146^ns-ko abnormal brain. (B) A Chi-square test was performed to compare the incidence of abnormal olfactory bulbs in WT and CD146^ns-ko groups.</p