Baseline characteristics between Baduanjin and control groups.

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

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

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

The chemical reagents used in this study.

<p>The chemical reagents used in this study.</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

Analysis of the enzymatic activity of deglycosylated DNase I and endo-1, 4-β-mannosidase obtained from co- and post-fermentation with Endo H-P.

<p>(A). M DNA molecular weight markers (the size of each band was indicated on the left); Lane 1 pHBM905A plasmid (about 300 ng); Lane 2 pHBM905A treated with 1 μl supernatant of DNase I; Lane 3–4 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with post-fermentation treatment; Lane 5 pHBM905A treated with 1 U commercial DNase I; Lane 6–7 pHBM905A treated with 0.5 and 1 μl of deglycosylated DNase I with co-fermentation treatment; Lane 8 pHBM905A treated with fermentation supernatant from <i>P</i>. <i>pastoris</i> bearing pHM905A plasmid (the negative control). (B). A hole of about 2mm was made with a hole puncher on a MD plate supplemented with 1% konjac powder and 0.05% trypan blue. The samples were added into the wells and the plate was incubated at 37°C, overnight. Sample 1: 1.5 μl supernatant of <i>P</i>. <i>pastoris</i> expressing glycosylated endo-1, 4-β-mannosidase; Sample 2: 2 μl supernatant of <i>P</i>. <i>pastoris</i> expressing glycosylated mannanase; Sample 3: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with post-fermentation treatment; Sample 4: 2 μl supernatant of deglycosylated endo-1, 4-β-mannosidase with co-fermentation treatment; Sample 5: 2 μl supernatant of EndoH-P.</p