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Glycogen synthase kinase-3β (GSK-3β) is required in the expression of epithelial junction proteins. It was found downregulated in hepatocellular carcinoma (HCC) tissues. The purpose of this study was to investigate the role of GSK-3β in modulating the metastatic behaviors of human HCC cell lines in vitro. In this study, the expression level of GSK-3β was measured in 4 human HCC cell lines, and the small interfering RNA (siRNA) vectors against or plasmids encoding GSK-3β were used to evaluate the responses of target cells to the knockdown or overexpression of this kinase, respectively. Our results showed that GSK-3β expression was significantly lower in human HCC cell lines with high metastatic potential than that in HCC cell lines without metastatic characteristics or in a normal human liver cell line. The knockdown of GSK-3β by siRNA led to a decreased expression of the epithelial junction molecules (ZO-1, E-cadherin) and an increase in the expression of a mesenchymal cell marker (α-SMA) and a gene transcription factor (β-catenin), resulting in enhanced tumor cell dissemination. In contrast, gain-of-function studies revealed that ectopic expression of GSK-3β reduced invasive and migratory abilities of HCC cells accompanied by decreased HCC cell proliferation and induced apoptosis. More importantly, downregulation of GSK-3β led to an increase in the expression and accumulation of β-catenin in the nuclei, promoting gene transcription. In conclusion, GSK-3β might play a vital role in suppressing HCC dissociation by preventing the disassembly of cancer cell epithelial junctional complex via the GSK-3β/β-catenin pathway.

Table S1. PCR primers and data analysis used in this study. (DOCX 50 kb)

Osteoporosis is closely associated with intervertebral disc degeneration. While parathyroid hormone (PTH) 1–34, which is an established drug used to treatosteoporosis, is thought to inhibit the disc degeneration associated with osteoporosis, the precise mechanism involved remains unclear. In the present study, primary Sprague-Dawley rat nucleus pulposus cells (NPCs) were cultured, phenotyped and then treated with dexamethasone (DXM) for 48 h. Cell area analysis and β-galactosidase staining were used to investigate the effect of DXM on the senescence of NPCs. In addition, the protein levels of LC3-II, Beclin-1, P62, p-mTOR and p-p70S6k were determined by western blotting and analyzing the regulatory effect of PTH upon autophagy and the mTOR signaling pathway in cells treated with DXM. Following autophagic inhibition induced by ATG5 siRNA transfection, the regulatory effect of PTH on senescence in NPCs were investigated in addition to the potential role of autophagy. As the concentration of DXM increased, the size of the NPCs was significantly enlarged and the proportion of cells with positive β-galactosidase staining increased significantly (P

Glycogen synthase kinase-3β (GSK-3β) is required in the expression of epithelial junction proteins. It was found downregulated in hepatocellular carcinoma (HCC) tissues. The purpose of this study was to investigate the role of GSK-3β in modulating the metastatic behaviors of human HCC cell lines in vitro. In this study, the expression level of GSK-3β was measured in 4 human HCC cell lines, and the small interfering RNA (siRNA) vectors against or plasmids encoding GSK-3β were used to evaluate the responses of target cells to the knockdown or overexpression of this kinase, respectively. Our results showed that GSK-3β expression was significantly lower in human HCC cell lines with high metastatic potential than that in HCC cell lines without metastatic characteristics or in a normal human liver cell line. The knockdown of GSK-3β by siRNA led to a decreased expression of the epithelial junction molecules (ZO-1, E-cadherin) and an increase in the expression of a mesenchymal cell marker (α-SMA) and a gene transcription factor (β-catenin), resulting in enhanced tumor cell dissemination. In contrast, gain-of-function studies revealed that ectopic expression of GSK-3β reduced invasive and migratory abilities of HCC cells accompanied by decreased HCC cell proliferation and induced apoptosis. More importantly, downregulation of GSK-3β led to an increase in the expression and accumulation of β-catenin in the nuclei, promoting gene transcription. In conclusion, GSK-3β might play a vital role in suppressing HCC dissociation by preventing the disassembly of cancer cell epithelial junctional complex via the GSK-3β/β-catenin pathway.

Highway maintenance equipment arrangement is a complex optimization problem. To solve this problem, a new “maintenance equipment service capacity (MESC)” index is introduced to evaluate the maintenance equipment service range. And an improved genetic algorithm based maintenance equipment arrangement method is put forward. Using this method, maintenance equipment can breakthrough the traditional work zone limit and the service range of maintenance equipment can be improved to the greatest extent. Practical experiments proved that the method in this paper can effectively reduce the redundant rate and improve equipment utilization of maintenance equipment arrangement.

Objective. To clarify the impact of H221Y mutation on drug resistance to NVP.Methods. 646 bp HIV-1polgene fragments (from 592 to 1237 nucleotide) with different NNRTIs mutation profiles from AIDS patients receiving antiretroviral therapy containing NVP regimens were introduced into pNL4-3 backbone plasmid. H221Y and (or) Y181C mutations were reverted to wild type amino acids by site-directed mutagenesis, then strains containing various mutation patterns were packaged. Phenotypic drug resistance was analyzed on TZM-bl cells.Results. 12 strains containing different drug-resistant mutation profiles were constructed, including the K101Q series (K101Q/Y181C/H221Y, K101Q/Y181C, K101Q/H221Y, and K101Q), the V179D series (V179D/Y181C/H221Y, V179D/Y181C, V179D/H221Y, and V179D), and the K103N series (K103N/Y181C/H221Y, K103N/Y181C, K103N/H221Y, K103N). For strains containing the mutation profiles (K101Q/Y181C, K101Q, V179D/Y181C, V179D, K103N/Y181C, and K103N), the presence of H221Y reduced NVP susceptibility by2.1±0.5to3.6±0.5fold. To the mutation profiles K101Q/H221Y, K101Q, V179D/H221Y, V179D, K103N/H221Y, and K103N, the presence of Y181C reduced NVP susceptibility by41.9±8.4to1297.0±289.1fold. For the strains containing K101Q, V179D, and K103N, the presence of Y181C/H221Y combination decreased NVP susceptibility by100.6±32.5to3444.6±834.5fold.Conclusion. On the bases of various NNRTIs mutation profiles, Y181C remarkably improved the IC50to NVP, although H221Ymutation alone just increases 2.1 ∼ 3.6-fold resistance to NVP, the mutation could improve 100.6 ∼ 3444.6-fold resistance to NVP when it copresent with Y181C, the phenotypic drug resistance fold was improved extremely. For strains containing the mutation profiles (K101Q/Y181C, K101Q, V179D/Y181C, V179D, K103N/Y181C, and K103N), the presence of H221Y reduced NVP susceptibility by2.1±0.5to3.6±0.5fold.

Pterostilbene (PTE), a natural plant extract, has an anti-inflammatory effect; however, whether PTE could protect nucleus pulposus cells (NPCs) in the intervertebral disk from inflammation remains unclear. Primary NPCs isolated from Sprague-Dawley (SD) rats were cultured, and Cell Counting Kit-8 (CCK-8) analysis was used to test the cytotoxicity of PTE. The effect of PTE on interleukin-1β (IL-1β)-induced inflammation was analyzed using an enzyme-linked immunosorbent assay, real-time polymerase chain reaction (PCR), and a Griess test. Western blotting, immunofluorescence, and a nuclear factor erythroid 2-related factor 2 (Nrf2) small interfering RNA (siRNA) transfection were used to assess the involvement of Nrf2 in the anti-inflammatory mechanism of PTE on NPCs. The results of the CCK-8 analysis showed that PTE produced no cytotoxicity in NPCs at 20 μM for 24 h. PTE suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2) and inhibited the messenger RNA (mRNA) expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) induced by IL-1β. PTE could promote the nuclear translocation of Nrf2 in NPCs. In addition, Nrf2 silence reversed the inhibitory effect of PTE on the production of NO and PGE2 and the expression of COX-2 and iNOS. These results indicate that PTE inhibits inflammation in the rat nucleus pulposus by promoting the nuclear translocation of Nrf2.

It is very important for the clinical management to test for minor HIV-1 resistance mutations accurately and sensitively. The conventional genotypic assays of HIV drug resistance detection based on sequencing can only discriminate the mutations which present in more than 20% - 30%. The aim of this study was to evaluate allele-specific real-time PCR (ASPCR) to detect the resistance-related mutations located at positions 103, 184 and 215.We developed the allele-specific PCR assay, using the most common drug resistance mutations in Chinese AIDS patients, K103N, M184V/I, T215F/Y as a model system. The standards were constructed by cloning the wild-type and mutant DNA fragments into the T-vector. We designed specific primers to discriminate mutant templates in the real-time PCR using SYBR green as a fluorescence reporter. And then we evaluated the ASPCR assay and tested 140 clinical samples using this method.The sensitivities of ASPCR assay were 0.04% for K103N, 0.30% for M184I, 0.40% for M184V, 0.03% for T215F and 0.02% for T215Y. The intra-assay and inter-assay coefficients of variation were less than 0.42. One hundred and forty plasma samples were tested by ASPCR and dynamic resistance curves of ten patients were obtained.Drug resistance emerged half a year after the start of antiretroviral therapy. The mutation of T215Y emerged 1 to 1.5 years after starting treatment and then increased rapidly. The ASPCR assay we developed was a sensitive, accurate and rapid method to detect the minor HIV-1 variants and it can provide earlier and more drug-resistance information for HIV research and AIDS antiretroviral therapy.

To elucidate the molecular evolutional characteristics of HIV-1 nucleoside reverse transcriptase inhibitor (NRTI) drug resistance-associated mutations in patients with AIDS receiving highly active antiretroviral therapy.We selected 4 AIDS patients receiving highly active antiretroviral therapy (HAART) with good adherence under a HIV-1 drug resistance cohort from a rural region in central China. Those people carried susceptible virus at the beginning of treatment and gradually came to produce virus resistant to NRTIs during the process of antiretroviral therapy (ART). Reverse transcriptase (RT) genes from each patient's peripheral blood samples (from 3 to 33 months after withdrawal) were cloned and sequenced in succession.We sequenced a total number of 855 clones and obtained the HIV-1 NRTI drug resistance-associated mutations patterns of the 4 patients. Typical resistance mutations of thymidine analogue mutations (TAMs) pattern 1, such as L210W, T215Y and M41L, were generated in patient 'A'. TAMs pattern 2, including D67N, K70R and K219Q mutations, was discovered in patient 'B'. Interestingly, in patient 'C', some clones comprising not only TAMs pattern 1 mutations (T215Y) but also TAMs pattern 2 mutations (K70R, D67N).The four patients show different pathways on HIV-1 NRTI drug resistance-associated mutations, including TAMs pattern 1, TAMs pattern 2 and the fusion pattern of TAMs-1TAMs-2. We also noticed that the tendency of gradual accumulation was obvious and those mutations detected earlier tended to be the predominant strains.