Community based Baduanjin exercise.

<p>Community based Baduanjin exercise.</p

Comparison of low score (<7) on SEMCD6 between two groups.

<p>Comparison of low score (<7) on SEMCD6 between two groups.</p

Baseline characteristics between Baduanjin and control groups.

<p>Baseline characteristics between Baduanjin and control groups.</p

SEMCD6 score before e vs. after intervention in Baduanjin group (x±δ).

<p>SEMCD6 score before e vs. after intervention in Baduanjin group (x±δ).</p

Flow of participants.

<p>Flow of participants.</p

Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in <i>E</i>. <i>coli</i>

<div><p>Ice nucleation protein (INP) is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N) to improve its surface display efficiency for human Arginase1 (ARG1). Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1) by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K₆-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K₆-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD₆₀₀ under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn²⁺, which could convert more than 95% of L-Arginine (L-Arg) to L-Ornithine (L-Orn) in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in <i>E</i>. <i>coli</i> cells.</p></div

Analyzing the characteristics of Endo H-P through mobility shift assay of RNase B.

<p>(A).Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 to Lane 6 denatured RNase B treated with concentrated Endo H-P at 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, respectively; Lane 7 denatured RNase B treated with concentrated Endo H-P at 37°C, respectively;Lane 8 the negative control (RNase B without treatment); Lane 9 the positive control (overdose of Endo H-P was added to the reaction system);(B). Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 the positive control (overdose of Endo H-P was added to the reaction system); Lane 2 the negative control (RNase B without treatment);Lane 3–8 denatured RNase B treated with Endo H-P at pH5.0, 5.5, 6.0, 6.5, 7.0 and 7.5, respectively.</p

Fluorescence microscope assay of InaK-N/ARG1s.

<p>Surface fluorescence of the cells harboring various InaK-N/ARG1s under a fluorescence microscope with the excitation laser of 488nm, and the EGFP detection channel being used. A-G indicated cells containing empty pET23a-T vector; pET23a-InaK-N/ARG1 vector; pET23a-ssMalE-InaK-N/ARG1 vector; pET23a-ssTorA-InaK-N/ARG1 vector; pET23a-D₆-InaK-N/ARG1 vector; pET23a-E₆-InaK-N/ARG1 vector; and pET23a-K₆-InaK-N/ARG1 vector, respectively. 1: detecting of FITC signal, 2: the bright field, 3: the merge of FITC signal and bright field.</p

Constructs in the study.

<p>(A) Different N-terminal fusions of InaK-N. (B) Different InaK-Ns fused with C-terminal human ARG1. (C) Schematic diagram of the cloning method for making the constructs. (D) <i>InaK-Ns</i> PCR products. Lanes 1–6 are PCR products for <i>InaK-N</i>, <i>ssMalE</i>-<i>InaK-N</i>, <i>ssTorA</i>-<i>InaK-N</i>, <i>D</i>_<i>6</i>-<i>InaK-N</i>, <i>E</i>_<i>6</i>-<i>InaK-N</i>, and <i>K</i>_<i>6</i>-<i>InaK-N</i>. (E) PCR products for <i>InaK-N/ARG1s</i>. Lane 1–8 <i>InaK-N</i>, <i>human ARG1</i>, <i>InaK-N/ARG1</i>, <i>ssMalE</i>-<i>InaK/ARG1</i>, <i>ssTorA</i>-<i>InaK-N/ARG1</i>, <i>D</i>_<i>6</i>-<i>InaK-N/ARG1</i>, <i>E</i>_<i>6</i>-<i>InaK-N/ARG1</i>, and <i>K</i>_<i>6</i>-<i>InaK-N/ARG1</i>. M: DNA Ladder.</p

Immobilization of the ARG1 fusions to the cell outer membrane.

<p>(A) SDS-PAGE and Western-Blot analysis of the human ARG1 fusions on the outer membrane. Upper panel is the SDS-PAGE analysis and the lower panel is the western-blot analysis. M: protein ladder; T: total cell proteins; OM: outer membrane proteins. InaK-N/ARG1; ssTorA-InaK-N/ARG1; K₆-InaK-N/ARG1; E₆-InaK-N/ARG1; D₆-InaK-N/ARG1; and ssMalE-InaK-N/ARG1 presented the recombinant strain expressing these fusion proteins, respectively; Control presented the recombinant strain containing empty pET23a-T. (B) Enzyme activity and proteinase K accessibility of the InaK-N/ARG1s. Proteinase K-: recombinant strain was untreated with proteinase K; Proteinase K+: recombinant strain was treated with proteinase K. K6-InaK-N/ARG1; E6-InaK-N/ARG1; D6-InaK-N/ARG1; ssMalE-InaK-N/ARG1; K6-InaK-N/ARG1 presented the recombinant strains expressing these fusion proteins, respectively.</p