Notch1 signaling contributes to TLR4-triggered NF-κB activation in macrophages.

Macrophages substantially influence the development, progression, and complications of inflammation-driven diseases. Although numerous studies support the critical role of Notch signaling in most inflammatory diseases, there is limited data on the role of Notch signaling in TLR4-induced macrophage activation and interaction of Notch signaling with other signaling pathways in macrophages during inflammation, such as the NF-κB pathway. This study confirmed that stimulation with lipopolysaccharide (LPS), a TLR4 ligand, upregulated Notch1 expression in monocyte/macrophage-like RAW264.7 cells and bone marrow-derived macrophages (BMDMs). LPS also induced increased mRNA expression of Notch target genes Notch1 and Hes1 in macrophages, suggesting that TLR4 signaling enhances activation of the Notch pathway. The upregulation of Notch1, Notch1 intracellular domain (NICD), and Hes1 proteins by LPS treatment was inhibited by DAPT, a Notch1 inhibitor. Additionally, the increased TNF-α, IL-6, and IL-1ß expression induced by LPS was inhibited by DAPT and rescued by jagged1, a Notch1 ligand. Furthermore, suppression of Notch signaling by DAPT upregulated Cylindromatosis (CYLD) expression but downregulated TRAF6 expression, IκB kinase (IKK) α/ß phosphorylation, and subsequently, phosphorylation and degradation of IκB-α, indicating that DAPT inhibited NF-κB activation triggered by TLR-4. Interestingly, DAPT did not inhibit the increased MyD88 expression induced by LPS. Our study findings demonstrate that macrophage stimulation via the TLR4 signaling cascade triggers activation of Notch1 signaling, which regulates the expression patterns of genes involved in pro-inflammatory responses by activating NF-κB. This effect may be dependent on the CYLD-TRAF6-IKK pathway. Thus, Notch1 signaling may provide a therapeutic target against infectious and inflammation-driven diseases.

Phytoremediation of Cadmium Contaminated Soil: Impacts on Morphological Traits, Proline Content and Stomata Parameters of Sweet Sorghum Seedlings.

Phytoremediation is a green, simple, eco-friendly, sustainable, and cost-effective remediation technology to remove and degrade contaminants from soil. In this study, a germination experiment and a pot experiment were performed in greenhouse to evaluate cadmium toxicity and phytoremediation capacity. The results showed that there was the highest membership function value of cadmium (MFVC) in KFJT-3 than that of KFJT-CK and KFJT-1, the value being 0.473, 0.456 and 0.413, respectively. Furthermore, the highest biomass was discovered in KFJT-3 compared to the other genotypes under 50 mg/kg cadmium stress. Physiological analysis showed that proline content significantly increased in KFJT-3, the value being 31.88%. In addition, Bioaccumulation factor (BAF) and Translocation factor (TF) value were 3.80 and 1.02 for KFJT-3, respectively. In conclusion, BAF and TF values showed that the cadmium tolerance of KFJT-1 and KFJT-3 could be higher than that of KFJT-CK, which could be the genotype for phytoremediation of cadmium contaminated soil.

Co-Culture of Adipose-Derived Stem Cells and Chondrocytes With Transforming Growth Factor-Beta 3 Promotes Chondrogenic Differentiation.

Tissue engineering cartilage is a promising strategy to reconstruct the craniofacial cartilaginous defects. It demands plenty of chondrocytes to generate human-sized craniofacial frameworks. Partly replacement of chondrocytes by adipose-derived stem cells (ADSCs) can be an alternative strategy.The study aimed at evaluating the chondrogenic outcome of ADSCs and chondrocytes in direct co-culture with transforming growth factor-beta (TGF-ß3). Porcine ADSCs and chondrocytes were obtained from abdominal wall and external ears. Four groups: ADSCs or chondrocytes monocultured in medium added with TGF-ß3; ADSCs and ACs co-cultured with or without TGF-ß3. Cell growth rate was performed to evaluate the cell proliferation. Morphological, histologic and real-time polymerase chain reaction analysis were performed to characterize the chondrogenic outcome of pellets. ADSCs had favorable multi-lineage differentiation potential. Further, when ADSCs were co-cultured with chondrocytes in medium added with TGF-ß3, the cell proliferation was promoted and the chondrogenic differentiation of ADSCs was enhanced. We demonstrate that pellet co-culture of ADSCs and chondrocyte with TGF-ß3 could construct high quantity cartilages. It suggests that this strategy might be useful in future cartilage repair.

A whole-transcriptome approach to evaluate reference genes for quantitative diurnal gene expression studies under natural field conditions in Tamarix ramosissima leaves

Background: Tamarix ramosissima is a desert forest tree species that is widely distributed in the drought-stricken areas to sustain the fragile ecosystem. Owing to its wide usage in the desert restoration of Asia, it can be used as an ecophysiological model plant. To obtain reliable and accurate results, a set of reference genes should be screened before gene expression. However, up to date, systematical evaluation of reference genes has not been conducted in T. ramosissima. Results: In this study, we used eigenvalues derived from principal component analysis to identify stable expressed genes from 72,035 unigenes from diurnal transcriptomes under natural field conditions. With combined criteria of read counts above 900 and CV of FPKM below 0.3, a total of 7385 unigenes could be qualified as candidate reference genes in T. ramosissima. By using three statistical algorithm packages, geNorm, NormFinder, and BestKeeper, the stabilities of these novel reference genes were further compared with a panel of traditional reference genes. The expression patterns of three aquaporins (AQPs) suggested that at least UBQ (high expression), EIF4A2 (low expression), and GAPDH (moderate expression) could be qualified as ideal reference genes in both RT-PCR and RNA-seq analysis of T. ramosissima. Conclusions: This work will not only facilitate future studies on gene expression and functional analysis of genetic resources of desert plants but also improve our understanding of the molecular regulation of water transport in this plant, which could provide a new clue to further investigate the drought adaptation mechanism of desert plant species under harsh environments.

The role of disulfide-bridge on the activities of H-shape gemini-like cationic lipid based siRNA delivery.

In our previous study, a H-shape gemini-like cationic lipid (ssGLCL, formerly named as CLD), composed of two hydrophilic lysine heads and two hydrophobic oleyl alcohol tails with a bridge of the redox-active disulfide-bond, had been synthesized and used as a nanocarrier for delivering small interfering RNAs (siRNAs) into cells. In order to further elucidate the role of disulfide (-S-S-) bridge on the activity of ssGLCL based siRNA delivery, a comparable ccGLCL bridged with a non-reducible carbon-carbon bond was synthesized and used as control in this study. Both two H-shape GLCL molecules could individually self-assemble into cationic nanoparticles in water phase and complex with negatively-charged siRNA into nanoplexes with particle size of ~200nm and zeta potential of ~ +30mV, and exhibit effective siRNA delivery both in vitro and in vivo. Investigation of internalization pathway displayed that both ssGLCL/siRNA and ccGLCL/siRNA nanoplexes were predominantly internalized into MCF-7 cells by the clathrin-mediated endocytosis pattern. Although a lower cellular uptake of siRNA was found in the human breast cancer MCF-7 cells, the ssGLCL/siRNA nanoplexes could exhibit similar or even stronger down-regulation effects on the targeted EGFR mRNA and protein in MCF-7 cells when compared to the ccGLCL/siRNA nanoplexes. Furthermore, mechanistic study showed that the enhanced down-regulation effects of ssGLCL/siRNA nanoplexes on targeted mRNA and protein were probably attributed to the increased release of siRNA from lysosomes to cytoplasm following the cleavage of redox-active disulfide-bridge in ssGLCL. Therefore, we believed that the redox-active H-shape ssGLCL could be a potential nanocarrier towards improving siRNA delivery.

Synergistic inhibition of breast cancer by co-delivery of VEGF siRNA and paclitaxel via vapreotide-modified core-shell nanoparticles.

A somatostatin analog, vapreotide (VAP), can be used as a ligand for targeting drug delivery based on its high affinity to somatostatin receptors (SSTRs), which is overexpressed in many tumor cells. RNA interference plays an important role on downregulation of vascular endothelial growth factor (VEGF), which is important for tumor growth, progression and metastasis. To improve tumor therapy efficacy, the vapreotide-modified core-shell type nanoparticles co-encapsulating VEGF targeted siRNA (siVEGF) and paclitaxel (PTX), termed as VAP-PLPC/siRNA NPs, were developed in this study. When targeted via somatostatin receptors to tumor cells, the VAP-PLPC/siRNA NPs could simultaneously delivery siVEGF and PTX into cells and achieve a synergistic inhibition of tumor growth. Interestingly, in vitro cell uptake and gene silencing experiments demonstrated that the targeted VAP-PLPC/siRNA NPs exhibited significant higher intracellular siRNA accumulation and VEGF downregulation in human breast cancer MCF-7 cells, compared to those of the non-targeted PEG-PLPC/siRNA NPs. More importantly, in vivo results further demonstrated that the targeted VAP-PLPC/siRNA NPs had significant stronger drug distribution in tumor tissues and tumor growth inhibition efficacy via receptor-mediated targeting delivery, accompany with an obvious inhibition of neovascularization induced by siVEGF silencing. These results suggested that the co-delivery of siRNA and paclitaxel via vapreotide-modified core-shell nanoparticles would be a promising approach for tumor targeted therapy.

Core-shell type lipid/rPAA-Chol polymer hybrid nanoparticles for in vivo siRNA delivery.

Our previous study had reported that cholesterol-grafted poly(amidoamine) (rPAA-Chol polymer) was able to self-assemble into cationic nanoparticles and act as a potential carrier for siRNA transfection. In this study, the core-shell type lipid/rPAA-Chol hybrid nanoparticles (PEG-LP/siRNA NPs and T7-LP/siRNA NPs) were developed for improving in vivo siRNA delivery by modifying the surface of rPAA-Chol/siRNA nanoplex core with a lipid shell, followed by post-insertion of polyethylene glycol phospholipid (DSPE-PEG) and/or peptide (HAIYPRH, named as T7) modified DSPE-PEG-T7. The integrative hybrid nanostructures of LP/siRNA NPs were evidenced by dynamic light scattering (DLS), confocal laser scanning microscope (CLSM), cryo-transmission electron microscope (Cryo-TEM) and surface plasmon resonance (SPR) assay. It was demonstrated that the T7 peptide modified LP/siRNA NPs (T7-LP/siRNA NPs) exhibited uniform and spherical structures with particle size of 99.39 ± 0.65 nm and surface potential of 42.53 ± 1.03 mV, and showed high cellular uptake efficiency and rapid endosomal/lysosomal escape ability in MCF-7 cells. Importantly, in vitro gene silencing experiment demonstrated that both of pegylated and targeted LP/siEGFR NPs exhibited significantly stronger downregulation of EGFR protein expression level in MCF-7 cells, compared to that of the physical mixture of siRNA lipoplexes and rPAA-Chol/siRNA nanoplexes. In vivo tumor therapy on nude mice bearing MCF-7 tumors further confirmed that the targeted T7-LP/siEGFR NPs exhibited the greatest inhibition on tumor growth via transferrin receptor-mediated targeting delivery, without any activation of immune responses and significant body weight loss following systemic administration. These findings indicated that the core-shell type T7-LP/siRNA nanoparticles would be promising siRNA delivery systems for in vivo tumor-targeted therapy.

Self-assembly cationic nanoparticles based on cholesterol-grafted bioreducible poly(amidoamine) for siRNA delivery.

In this study, a series of bioreducible poly(amidoamine)s grafting different percentages of cholesterol (rPAA-Ch14: 14%, rPAA-Ch29: 29%, rPAA-Ch57: 57% and rPAA-Ch87: 87%) was synthesized and used for siRNA delivery. These amphiphilic polymers were able to self-assemble into cationic nanoparticles in aqueous solution at low concentrations. The nanoparticle formation was evidenced via cryo-transmission electron microscope (Cryo-TEM) and dynamic light scattering analysis. The average hydrodynamic size of rPAA-Ch blank nanoparticles was about 80-160 nm with zeta potential of 50-60 mV. Also, the effects of different percentages of cholesterol grafted onto rPAA on physicochemical characteristics, in vitro cytotoxicity, cellular uptake, VEGF gene silencing efficacy and translocation mechanism of rPAA-Ch/siRNA complexes were investigated. The results showed that rPAA-Ch57 polymer was not only able to form stable nanocomplexes and possess high cell uptake, but also to exhibit the best in vitro VEGF gene silencing efficacy and the best in vivo tumor growth inhibition effect when it was formulated with VEGF-siRNA. Moreover, the observations of confocal laser scanning microscope (CLSM) and the study of cholesterol competitive inhibition demonstrated that endosomal/lysosomal escape and cytoplasmic dissociation of rPAA-Ch57/siRNA complexes were dependent on the "proton sponge effect" and disulfide cleavage, following internalization with cholesterol-related endocytosis pathway and subsequent transportion into endosomes/lysosomes. These findings indicated that the rPAA-Ch57 polymer should be a promising and potent carrier for siRNA delivery.

Isoliquiritigenin enhances radiosensitivity of HepG2 cells via disturbance of redox status.

Redox balance plays an important role in the maintenance of cell growth and survival. Disturbance of this equilibrium can alter normal cellular processes. Excessive reactive oxygen species (ROS) are often found in cancer cells. However, cancer cells have an efficient antioxidant system to counteract the increased generation of ROS. This high antioxidant capacity also favors resistance to drugs and radiation. Here, we show that isoliquiritigenin (ISL), a natural antioxidant, effectively decreased ROS in HepG2 cells in a time-dependant manner at 0.5, 1, and 2 h of treatment. The decreased ROS caused redox imbalance and reductive stress. To adapt to this state, nuclear factor erythroid-2-related factor 2, which regulates the antioxidant enzyme system, was significantly decreased. Antioxidant enzymes reached their lowest level at 6 h after ISL treatment. Endogenous ROS were still being generated so after 6 h of ISL treatment, ROS were clearly higher than before ISL treatment, causing redox imbalance in the HepG2 cells which changed from reductive to oxidative stress. At this stage, cells were irradiated with X-rays. The excess ROS induced serious oxidative stress, resulting in radiosensitization. Therefore, we concluded that ISL induced oxidative stress by disturbing the redox status and ultimately enhancing the radiosensitivity of HepG2 cells.

Effects of carbon-ion beam or X-ray irradiation on anti-apoptosis ΔNp73 expression in HeLa cells.

ΔNp73 has emerged as an interesting novel factor in cancer research. Here, we report the effect of carbon-ion beams on ΔNp73 expression in human cervix carcinoma HeLa cells in contrast to the effect of X-rays. Cellular sensitivities were determined by colony formation. Radiation-induced cell cycle arrest was investigated with flow cytometry. Additionally, radiation-induced apoptosis was analyzed with flow cytometry and Hoechst staining. Furthermore, ΔNp73 expression was examined by semi-quantitative reverse transcription-PCR (semi-quantitative RT-PCR) as well as by Western blot analysis. Following irradiation, stronger G2/M phase arrest, more significant increase in apoptosis and more pronounced ΔNp73 degradation were observed after exposure to high-LET carbon beams in comparison with X-rays at 4 Gy doses. These observations indicate that there is a differential ΔNp73 expression in response to different LET radiations, and down-regulated ΔNp73 expression might play a critical role in promoting cycle arrest and apoptosis in cancer cells. This study highlights the potential of ΔNp73 in radiotherapy.

[The morphological study on masseter muscle following mandibular angle osteotomy].

OBJECTIVE: To evaluate the morphological change of masseter after the mandibular angle osteotomy. METHODS: Computerized tomography (CT) examination was performed on 120 patients treated by mandibular angle osteotomy before operation and at 3, 6, 12 months after operation, respectively. The pre- and postoperative masseter muscle thickness and cross-sectional area were evaluated using 3D CT images observed from 3 selected slice planes, which were paralleled with Frankfurt horizontal plane. These CT images were stored and three-dimensional reconstruction were made for calculation of masseter muscle volume through software. RESULTS: After operation, the reduction of the masseter muscle volume and cross-sectional area was seen. The volume of the masseter at 3, 6, 12 months postoperatively decreased to 82.02%, 77.00% and 80.43% (P < 0.05). The cross-sectional area at 3, 6,12 months postoperatively decreased to 85.81%, 78.86% and 81.56% at A plane, 80.94%, 75.03% and 77.04% at B plane, and reached to 13.46%, 11.48% and 13.89% at C plane (P < 0.05, P < 0.05, P < 0.01). The masseter thickness after operation was significantly different from that before operation during the follow-up period, but not at 12 months after operation at A plane. CONCLUSIONS: The masseter atrophy happens spontaneously after mandibular angle osteotomy, especially at the region of mandibular angle. It should be considered during surgical design.

[Effects of cembrane-type diterpenes on proliferation of PC12 cells and their antagonistic effects on neurotoxicity induced by glutamate].

OBJECTIVE: To investigate the effects of cembrane-type diterpenes extracted from Sinularia flexibilis on the proliferation of PC12 cells and their protective effects on PC12 cells exposed to glutamate. METHODS: Methyl thiazolyl tetrazolium (MTT) method was adopted to observe the effects of cembrane-type diterpenes (compound 1, compound 2 and compound 3) on the proliferation of PC12 cells. And the protective effects of the three compounds on PC12 cells exposed to glutamate were also detected by MTT. Furthermore, the influence of compound 1 on intracellular concentration of calcium in PC12 cells exposed to glutamate was detected by laser confocal microscopy. RESULTS: After 72-hour PC12 cell culture, OD values in the 2, 10 and 50 micromol/L compound 1 groups were significantly higher than that in the normal group (P<0.05, P<0.01). After 24-hour glutamate damage, OD values in the 0.4, 2 and 50 micromol/L compound 1 groups, the 0.4, 2 and 100 micromol/L compound 2 groups and the 2 micromol/L compound 3 group were obviously increased as compared with the untreated group (P<0.01, P<0.05). After 48-hour glutamate damage, OD values in the compound 1 group were approximate to those in the normal control and the positive control group while were significantly higher than that in the untreated group (P<0.01, P<0.05), but no dose-dependent effect was observed. Compound 1 of 0.4, 2, 50 micromol/L could significantly reduce the intracellular concentration of calcium in PC12 cells exposed to glutamate (P<0.05, P<0.01), which was also approximate to the effect of nimodipine (positive control drug). CONCLUSION: Cembrane-type diterpenes (compound 1, compound 2 and compound 3) extracted from Sinularia flexibilis have obvious protective effects on PC12 cells damaged by glutamate, and compound 1 has the best neuroprotective effect. The mechanism of the neuroprotective effect of compound 1 may lie in reducing the intracellular concentration of calcium in PC12 cells exposed to glutamate and relieving the calcium overload.

Intragenomic and interspecific 5S rDNA sequence variation in five Asian pines.

Patterns of intragenomic and interspecific variation of 5S rDNA in Pinus (Pinaceae) were studied by cloning and sequencing multiple 5S rDNA repeats from individual trees. Five pines, from both subgenera, Pinus and Strobus, were selected. The 5S rDNA repeat in pines has a conserved 120-base pair (bp) transcribed region and an intergenic spacer region of variable length (382-608 bp). The evolutionary rate in the spacer region is three- to sevenfold higher than in the genic region. We found substantial sequence divergence between the two subgenera. Intragenomic sequence heterogeneity was high for all species, and more than 86% of the clones within each individual were unique. The 5S gene tree revealed that different 5S repeats within individuals are polyphyletic, indicating that their ancestral divergence preceded the speciation events. The degrees of interspecific and intragenomic divergence among diploxylon pines are similar. The observed sequence patterns suggest that concerted evolution has been acting after the diversification of the two subgenera but very weak after the speciation of the four diploxylon pines. Sequence patterns in P. densata are consistent with hybrid origin. It had higher intragenomic diversity and maintained polymorphic copies of the parental types in addition to new and recombinant types unique to the hybrid.