Fibroblast spheroids were reseeded on TCPS after chitosan treatment.

<p>(a) SA-β-gal staining of PD48-Cd3-PD2 cells. Scale bar = 200 μm (b) The percentage of PD50 and PD48-Cd3-PD2 cells stained for SA-β-gal. At least 400 cells were calculated from ten randomly selected fields for each case. **p<0.01. (c) BrdU incorporation assay of PD50 and PD48-Cd3-PD2 cells (n = 4). *p<0.05. (d) Cell cycle (PI staining) analysis of PD52 and PD48-Cd3-PD2 cells. (e) The PD curve with and without chitosan treatment (n = 3). Red arrows indicate PD48 (1st treatment) and PD70 (2nd treatment) cells were seeded on chitosan for 3 days and reseeded on TCPS for serial passages.</p

Fibroblasts sorted by FSC and FITC.

<p>(a) The gating range of cell sorting. P1: PD45 (+, +) population. P2: PD45 (-, -) population. (b) Viability of PD45 (+, +) cells determined by PI uptake and annexin-V-FITC labeling. (c) SA-β-gal staining of PD45 (+, +), PD45 (-, -), PD45 (+, +)-Cd3, and PD45 (-, -)-Cd3 cells. Scale bar = 200 μm (d) The percentage of PD45 (+, +), PD45 (-, -), PD45 (+, +)-Cd3, and PD45 (-, -)-Cd3 cells stained for SA-β-gal. At least 400 cells were calculated from ten randomly selected fields for each case. **p<0.01. (e) BrdU incorporation assay of PD45 (+, +), PD45 (-, -), PD45 (+, +)-Cd3, and PD45 (-, -)-Cd3 cells (n = 4). *p<0.05.</p

Chitosan Treatment Delays the Induction of Senescence in Human Foreskin Fibroblast Strains

<div><p>Fibroblasts have been extensively used as a model to study cellular senescence. The purpose of this study was to investigate whether the human foreskin fibroblast aging process could be regulated by using the biomaterial chitosan. Fibroblasts cultured on commercial tissue culture polystyrene (TCPS) entered senescence after 55–60 population doublings (PDs), and were accompanied by larger cell shape, higher senescence-associated β-galactosidase (SA β-gal) activity, lower proliferation capacity, and upregulation of senescence-associated molecular markers p21, p53, retinoblastoma (pRB), and p16. Before senescence was reached, PD48 cells were collected from TCPS and seeded on chitosan for three days (PD48-Cd3) to form multicellular spheroids. The protein expression of senescence-associated secretory phenotypes (SASPs) and senescence-associated molecular markers of these cells in PD48-Cd3 spheroids were downregulated significantly. Following chitosan treatment, fibroblasts reseeded on TCPS showed lower SA β-gal activity, increased cellular motility, and a higher proliferation ability of 70–75 PDs. These phenotypic changes were not accompanied by colonies forming in soft agar and a continuous decrease in the senescence-associated proteins p53 and pRB which act as a barrier to tumorigenesis. These results demonstrate that chitosan treatment could delay the induction of senescence which may be useful and safe for future tissue engineering applications.</p></div

Effect of chitosan treatment on scratch wound assay.

<p>(a) Both wounds were gradually filled with PD50 and PD48-Cd3-PD2 cells. (b) The percentage of wound closure by PD50 and PD48-Cd3-PD2 cells (n = 5). **p<0.01.</p

The characteristics of fibroblasts after chitosan treatment.

<p>(a) Western blot results of senescence-associated protein pRB, p16, p21 and p53 expression in PD20, PD50, and PD48-Cd3-PD2 cells. (b) Colony formation of HeLa and PD45-Cd3-PD2 cells by soft agar assay.</p

The characteristics of fibroblast spheroids after chitosan treatment.

<p>(a) Western blot results of proteins pRB, p16, p21, p53, TGF-β, IL-1β, IL-6 and IL-8 expression in PD48 and PD48-Cd3 cells. (b) The relative amount of protein pRB, p16, p21, p53, TGF-β, IL-1β, IL-6 and IL-8 expression in PD48 and PD48-Cd3 cells. (*p<0.05 and **p<0.01, n = 5). (c) BrdU incorporation assay of PD10, PD48, PD48-Cd3, and H₂O₂-treated cells (**p<0.01, n = 5).</p

Freestanding Cathode Electrode Design for High-Performance Sodium Dual-Ion Battery

In this work, both advantages of sodium-ion batteries and dual-ion batteries have been combined in an innovated sodium-ion-based dual-ion battery (SDI) system using a Na metal film as anode and a freestanding meso-carbon microbead film (FS-MCMB) as cathode. FS-MCMB in SDI battery exhibited a superior working performance with the specific capacity of 83.6 mAh/g and a remarkable long-term stability over 300 cycles. The SDI battery with FS-MCMB exhibited an advantage of high mass density loading in the range of 2–7.5 mg cm^–2, which was equal to a comparable capacity of 78–83 mAh/g. The electrochemical impedance analysis indicated that FS-MCMB provided a superior permeability, resulting in facilitating electrolyte infiltration into MCMB structure. In-situ XRD and ex-situ Raman spectroscopy were utilized to characterize the intercalating/deintercalating process of PF₆^– anions into/out of MCMB during charging/discharging processes. Finally, theoretical calculations further confirmed the structural arrangement of PF₆^– anions in the graphite layers

Subcellular localization of JEV NS5.

<p>(A-C) Cellular lysates of HEK293T cells infected with JEV (MOI = 5) or transfected with NS5-Flag for 24 h underwent Qproteome Mitochondria Isolation (A) or biochemical fractionation as outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004750#ppat.1004750.s005" target="_blank">S5B and S5C Fig</a>, respectively (B and C). Western blot analysis of indicated proteins in cytosolic and crude mitochondrial fractions. C, cytosolic fraction; H, heavy membrane fraction/crude mitochondrial fraction; L, light microsomal membrane fraction. (D) The crude mitochondrial fraction isolated from HEK293T cells infected with JEV (MOI = 3) or transfected with NS5-Flag for 24 h was treated with Proteinase K (100 μg/ml) for 30 min on ice. The reactants were developed by Western blot analysis with antibodies against NS5 or the indicated mitochondrial proteins. (E) Confocal microscopy of pEYFP-Mito-NS5-A549 cells stained with anti-Flag plus Alexa Fluor 568 goat anti-rabbit and anti-HADHα plus Alexa Fluor 647 goat anti-mouse antibody. (F) Confocal microscopy of pEYFP-Mito-NS5-A549 cells transfected with HADHβ-HA for 24 h and stained with anti-Flag plus Alexa Fluor 568 goat anti-rabbit and anti-HA plus Alexa Fluor 647 goat anti-mouse antibody.</p

The recombinant JEV with NS5-M19A mutation is less able to block LCFA β-oxidation and induces less cytokine expression.

<p>(A and B) A549 cells infected with JEV-WT or JEV-NS5-M19A (MOI = 10) for 5 h were changed to serum-free medium for 1 h, then incubated with PA-BSA or BSA control. (A) Real-time OCR was measured from 6 to 24 h post-infection. The OCR before PA-BSA or BSA treatment was set to 100%. (B) The AUC OCR with PA-BSA and BSA (n = 3). (C and D) A549 cells infected with the indicated JEV (MOI = 10) for 5 h were incubated with serum-free medium for 1 h before treatment with PA-BSA or BSA for 18 h. RT-qPCR analysis of relative mRNA levels of IL-6 (C) and TNF-α (D) (n = 3). (E-G) A549 cells were infected with JEV-WT or JEV-NS5-M19A (MOI = 10) for 24 h in serum (10% FBS)-containing medium. RT-qPCR analysis of relative mRNA levels of JEV RNA (E), IL-6 (F) and TNF-α (G) (n = 3). Data are mean±SD.*P < 0.05, **P < 0.01 and ***P < 0.001.</p

Reduced neurovirulence of NS5-M19A—mutated JEV in challenged mice.

<p>(A) Survival in C57BL/6 mice infected with 0.2, 2 or 20 plaque-forming units (PFU) of JEV-WT or JEV-NS5-M19A by an intracerebral (i.c.) injection. The animal number (<i>n</i>) and survival rate for each group are shown. (B-D) RT-qPCR of relative JEV RNA (B), IL-6 (C), and TNF-α (D) mRNA levels in brain tissues of mice inoculated with JEV-WT or JEV-NS5-M19A (20 PFU) (n = 3). Data are mean±SD.*P < 0.05.</p