GSK3 Inhibitor-BIO Regulates Proliferation of Immortalized Pancreatic Mesenchymal Stem Cells (iPMSCs)

<div><h3>Background</h3><p>The small molecule 6-bromoindirubin-30-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, is a pharmacological agent known to maintain self-renewal in human and mouse embryonic stem cells (ESCs). However, the precise role of GSK3 in immortalized pancreatic mesenchymal stem cells (iPMSCs) growth and survival is not completely understood at present.</p> <h3>Results</h3><p>To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effect of BIO on iPMSCs. We found that the inactivation of GSK3 by BIO can robustly stimulate iPMSCs proliferation and mass formation as shown by QRT-PCR, western blotting, 5-Bromo-2-deoxyuridine (BrdU) immunostaining assay and tunel assay. However, we did not find the related roles of BIO on β cell differentiation by immunostaining, QRT-PCR assay, glucose-stimulated insulin release and C-peptide content analysis.</p> <h3>Conclusions</h3><p>These results suggest that BIO plays a key role in the regulation of cell mass proliferation and maintenance of the undifferentiated state of iPMSCs.</p> </div

Therapeutic Protection Against H. pylori Infection in Mongolian Gerbils by Oral Immunization With a Tetravalent Epitope-Based Vaccine With Polysaccharide Adjuvant

Urease is an effective target for design of a therapeutic epitope vaccine against Helicobacter pylori (H. pylori). In our previous studies, an epitope vaccine CTB-UE containing Th and B epitopes from H. pylori urease was constructed, and the CTB-UE vaccine could provide therapeutic effect on H. pylori infection in mice. However, a multivalent vaccine, combining different antigens participating in different aspects of H. pylori colonization and pathogenesis, may be more effective as a therapeutic vaccine than a univalent vaccine targetting urease. Therefore, a multivalent epitope vaccine FVpE, containing Th1-type immune adjuvant NAP, three selected functional fragments from CagA and VacA, and an urease multi-epitope peptide (UE) from CTB-UE, was constructed in this study and expected to obtain better sterilizing immunity than the univalent epitope vaccine CTB-UE. The therapeutic effect of multivalent epitope vaccine FVpE with polysaccharide adjuvant (PA) was evaluated in H. pylori-infected Mongolian gerbil model. The results showed that both FvpE and CTB-UE vaccine could induce similar levels of specific antibodies against H. pylori urease, and had similar inhibition effect on H. pylori urease activity. However, only FVpE could induce high levels of specific antibodies to CagA, VacA, and NAP. In addition, oral therapeutic immunization with FVpE plus PA significantly reduced the number of H. pylori colonies in the stomach of Mongolian gerbils compared with oral immunization with CTB-UE plus PA, or FVpE only, and the FVpE vaccine with PA even exhibited sterilizing immunity. The protection of FVpE was related to the mixed CD4+ T cell responses and epitope-specific antibodies against various H. pylori antigens. These results indicate that a multivalent epitope vaccine targetting various H. pylori antigens could be a promising candidate against H. pylori infection

Effects of Al on Precipitation Behavior of Ti-Nb-Ta-Zr Refractory High Entropy Alloys

Addition of Al can decrease density and improve oxidation resistance of refractory high entropy alloys (RHEAs), but may cause complicated precipitation and further affect mechanical properties. The present work studied the microstructural evolution of Al-contained RHEAs at elevated temperatures. The effects of Al on precipitation behavior were discussed. Results show that, TiNbTa0.5ZrAlx alloys (x ≤ 0.5) have single BCC (Body Centered Cubic) structure, but the primary BCC phase is supersaturated. Precipitation of BCC2(Nb,Ta)-rich solid solution phase, HCP(Zr,Al)-rich intermetallic phase, and ordered B2 phase can occur during heat treatment at 600~1200 °C. The precipitation of BCC2 phase mainly exists in RHEAs with low content of Al, while HCP (Hexagonal Close Packed) precipitates prefer to form in RHEAs with high content of Al. Interestingly, ordered B2 precipitates with fine and basket-weave structure can form in TiNbTa0.5ZrAl0.5 alloy after annealing at 800 °C, producing significant precipitation hardening effect

Hepatoprotective Effects of Kaempferol-3-O-α-l-Arabinopyranosyl-7-O-α-l-Rhamnopyranoside on d-Galactosamine and Lipopolysaccharide Caused Hepatic Failure in Mice

Fulminant hepatic failure (FHF), associated with high mortality, is characterized by extensive death of hepatocytes and hepatic dysfunction. There is no effective treatment for FHF. Several studies have indicated that flavonoids can protect the liver from different factor-induced injury. Previously, we found that the extracts of Elaeagnus mollis leaves had favorable protective effects on acute liver injury. However, the role and mechanisms behind that was elusive. This study examined the hepatoprotective mechanisms of kaempferol-3-O-α-l-arabinopyranosyl-7-O-α-l-rhamnopyra-noside (KAR), a major flavonol glycoside of E. mollis, against d-galactosamine (GalN) and lipopolysaccharide (LPS)-induced hepatic failure. KAR reduces the mouse mortality, protects the normal liver structure, inhibits the serum aspartate aminotransferase (AST) and alamine aminotransferase (ALT) activity and decreases the production of malondialdehyde (MDA) and reactive oxygen species (ROS) and inflammatory cytokines, TNF-α, IL-6, and IL-1β. Furthermore, KAR inhibits the apoptosis of hepatocytes and reduces the expression of TLR4 and NF-κB signaling pathway-related proteins induced by GalN/LPS treatment. These findings suggest that the anti-oxidative, anti-inflammatory, and anti-apoptotic effects of KAR on GalN/LPS-induced acute liver injury were performed through down-regulating the activity of the TLR4 and NF-κB signaling pathways

Intracellular BCG survival analyzed by qPCR and CFU assay.

<p><b>(A)</b> The RAW264.7 cells were transfected with miR-144-3p control, miR-144-3p mimic or inhibitor, or were treated with rapamycin or 3-MA, and then infected with BCG for 24 h. The DNA of intracellular BCG was determined by using a qPCR assay. Cells trated with rapamycin or 3-MA were used as a positive or negtive control of autophagy. *p<0.05, **p<0.01, ***p<0.001. <b>(B)</b> The RAW264.7 cells were transfected with miR-144-3p control, miR-144-3p mimic or miR-144-3p inhibitor, or were treated with rapamycin or 3-MA. After that, the RAW264.7 cells were infected with BCG for 24 h, 48 h and 72 h. Intracellular BCG survival was evaluated by CFU assay. *p<0.05, **p<0.01, ***p<0.001.</p

MicroRNA-144-3p inhibits autophagy activation and enhances Bacillus Calmette-Guérin infection by targeting ATG4a in RAW264.7 macrophage cells

<div><p>MicroRNAs (miRNAs) are small noncoding nucleotides that play major roles in the response of host immune cells. Autophagy plays a key role in activating the antimicrobial host defense against <i>Mycobacterium tuberculosis</i> (<i>M</i>. <i>tuberculosis</i>). Whether miRNAs specifically influence the activation of macrophage autophagy during <i>M</i>. <i>tuberculosis</i> infection is largely unknown. In the present study, we demonstrate that <i>Mycobacterium bovis</i> Bacillus Calmette-Guérin (BCG) infection of macrophages leads to increased expression of miR-144-3p, which targets autophagy-related gene 4a (ATG4a), to inhibit autophagy activation and antimicrobial responses to BCG. Overexpression of miR-144-3p significantly decreased both mRNA and protein levels of ATG4a, inhibited the formation of autophagosomes in RAW264.7 cells and increased intracellular survival of BCG. However, transfection with miR-144-3p inhibitor led to an increase in ATG4a levels, accelerated the autophagic response in macrophages, and decreased BCG survival in macrophages. The experimental results of this study reveal a novel role of miR-144-3p in inhibiting autophagy activation by targeting ATG4a and enhancing BCG infection, and provide potential targets for developing improved treatment.</p></div

Tunel staining analysis iPMSCs cultured with BIO or not.

<p>Bar = 20 µm, **P<0.01.</p

miR-144-3p inhibited autophagosome formation.

<p><b>(A and B)</b> Transmission electron microscopy confirms repression of autophagy by miR-144-3p overexpression or knockdown. RAW264.7 cells were transfected with miR-144-3p control, miR-144-3p mimic or miR-144-3p inhibitor for 24 h, and then infected with BCG for 24 h. M, mitochondrion. Autophagosomes (denoted by red arrows). The number of autophagosomes per cross-sectioned cell was counted (15 cells per group counted by TEM). *p<0.05, **p<0.01. <b>(C and D)</b> Overexpression of miR-144-3p could attenuate induction of autophagy by rapamycin. The BCG-challenged RAW264.7 cells were transfected with miR-144-3p mimics, and then treated with 50 μg/ml rapamycin for 2 h. Meanwhile the BCG-challenged RAW264.7 cells were treated with rapamycin or 0.1% DMSO as controls. BCG bacteria (denoted by black arrows). M, mitochondrion. Autophagosomes (denoted by red arrows). The number of autophagosomes per cross-sectioned cell was counted (15 cells per group counted by TEM). **p<0.01, ***p<0.001.</p

β-catenin and BrdU incorporation assay.

<p>A, The mitiosis index with BIO was significantly higher than without BIO medium; B, The percentage of positive cells treated with BIO or not from A; C, BIO treatment caused nuclear accumulation of β-catenin, which was not observed in control cells; D, Cells were assayed for β-catenin-mediated transcriptional activation by using a reporter containing either functional (TOP-Flash) or mutated (FOP-Flash) Tcf binding sites. Cells stimulated with BIO showed rise in the levels of β-catenin activity, as measured by luciferase activity. Treatment with BIO resulted in the higher fold change (almost 4 fold) when TOP-Flash activity was normalized against FOP-Flash activity. There was no significant difference in activities in the presence of the mutated FOP-Flash reporter; C, Bar = 20 µm, *P<0.05; E, Western blotting showed that p-β-catenin (S33/37/T41) was decreased in BIO treated.</p