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The distribution of titanium (Ti) active sites in the framework of TS-1 zeolite is one of the critical factors affecting its catalytic performance, since it can influence accessibility of the Ti active sites and the catalytic utilization of Ti atoms. Here, TS-1 zeolite with homogeneous distribution of titanium atoms in the framework (named BUS-TS-1) is successfully synthesized via bottom-up strategy at high basicity conditions, and BUS-TS-1 zeolite exhibits better catalytic performance for both olefin and alkane oxidation. The comprehensive explorations on the crystallization behavior of BUS-TS-1 zeolite show that, the gripper-like silicon species, as condensed matter in Q2 and Q3 states (detected by 29Si NMR), can be obtained by the condensation of tetraethyl orthosilicate (TEOS) at high alkalinity conditions. The gripper-like silicon species can condense with Ti-OH to form stable and isolated Si-O-Ti species, so that the phase transfer rates of Si and Ti species match during the entire crystallization process, resulting in the homogeneous distribution of Ti atoms. This work offers a useful strategy for the synthesis of high-performance catalysts by regulating the distribution of Ti active-site within TS-1 framework.

There is growing interest in expanding the production of soybean oils (mainly triacylglycerol, or TAG) to meet rising feed demand and address global energy concerns. We report that a plastid-localized glycerol-3-phosphate dehydrogenase (GPDH), encoded by GmGPDHp1 gene, catalyzes the formation of glycerol-3-phosphate (G3P), an obligate substrate required for TAG biosynthesis. Overexpression of GmGPDHp1 increases soybean seed oil content with high levels of unsaturated fatty acids (FAs), especially oleic acid (C18:1), without detectably affecting growth or seed protein content or seed weight. Based on the lipidomic analyses, we found that the increase in G3P content led to an elevated diacylglycerol (DAG) pool, in which the Kennedy pathway-derived DAG was mostly increased, followed by PC-derived DAG, thereby promoting the synthesis of TAG containing relatively high proportion of C18:1. The increased G3P levels induced several transcriptional alterations of genes involved in the glycerolipid pathways. In particular, genes encoding the enzymes responsible for de novo glycerolipid synthesis were largely upregulated in the transgenic lines, in-line with the identified biochemical phenotype. These results reveal a key role for GmGPDHp1-mediated G3P metabolism in enhancing TAG synthesis and demonstrate a strategy to modify the FA compositions of soybean oils for improved nutrition and biofuel.

Brassica napus is an important oilseed crop in the world and the mechanism of seed oil biosynthesis in B. napus remains unclear. In order to study the mechanism of oil biosynthesis and generate germplasms for breeding, an EMS mutant population with ~100,000 M2 lines was generated using Zhongshuang 11 as the parent line. The EMS-induced genome-wide mutations in M2-M4 plants were assessed. The average number of mutations including single nucleotide polymorphisms and insertion/deletion in M2-M4 was 21,177, 28,675 and 17,915, respectively. The effects of the mutations on gene function were predicted in M2-M4 mutants, respectively. We screened the seeds from 98,113 M2 lines and 9,415 seed oil content and fatty acid mutants were identified. We further confirmed 686 mutants with altered seed oil content and fatty acid in advanced generation (M4 seeds). Five representative M4 mutants with increased oleic acid were re-sequenced and the potential causal variations in FAD2 and ROD1 genes were identified. This study generated and screened a large scale of B. napus EMS mutant population and the identified mutants could provide useful genetic resources for the study of oil biosynthesis and genetic improvement of seed oil content and fatty acid composition of B. napus in the future. Accepted version This research was supported by The National Key Research and Development Program of China (2016YFD0101000) and the National Natural Science Foundation of China (31871658).

Plant oils are valuable commodities for food, feed, renewable industrial feedstocks and biofuels. To increase vegetable oil production, here we show that the nonspecific phospholipase C6 (NPC6) promotes seed oil production in the Brassicaceae seed oil species Arabidopsis, Camelina and oilseed rape. Overexpression of NPC6 increased seed oil content, seed weight and oil yield both in Arabidopsis and Camelina, whereas knockout of NPC6 decreased seed oil content and seed size. NPC6 is associated with the chloroplasts and microsomal membranes, and hydrolyzes phosphatidylcholine and galactolipids to produce diacylglycerol. Knockout and overexpression of NPC6 decreased and increased, respectively, the flux of fatty acids from phospholipids and galactolipids into triacylglycerol production. Candidate-gene association study in oilseed rape indicates that only BnNPC6.C01 of the four homeologues NPC6s is associated with seed oil content and yield. Haplotypic analysis indicates that the BnNPC6.C01 favorable haplotype can increase both seed oil content and seed yield. These results indicate that NPC6 promotes membrane glycerolipid turnover to accumulate TAG production in oil seeds and that NPC6 has a great application potential for oil yield improvement.

The heterologous biosynthesis of complex natural products has enabled access to polyketide, nonribosomal peptide, isoprenoid, and other compounds with wide-spanning societal value. Though several surrogate host systems exist, Escherichia coli is often a preferred choice due to its rapid growth kinetics and extensive molecular biology protocols. However, a persistent challenge to the utilization of E. coli has been the successful in vivo reconstitution of type II polyketide synthase (PKS) systems. In particular, gene expression of the ketosynthase (KS) components of the minimal PKS has consistently yielded insoluble protein products. In the following report, two type II PKS systems were functionally reconstituted in E. coli. The approach to do so relied upon the utilization of the native transcriptional coupling between the dimeric KS subunits, leading to soluble recombinant protein products and successful polyketide biosynthesis. Resulting strains produced 10 mg/L TW95c and 25 mg/L dehydrorabelomycin. Hence, the strategy offers a new option in the biosynthetic engineering efforts for the heterologous production of type II polyketide products using E. coli.

The abnormal proliferation and apoptosis of human periodontal ligament stem cells (hPDLSCs) serves a crucial role in the development of periodontitis. Oroxylin A has shown protective effects in a variety of inflammatory diseases. The present study was aimed to investigate the effects of oroxylin A on lipopolysaccharide (LPS) treated hPDLSCs. In the present study, cells were exposed to different concentrations (10, 20, 40 uM) of oroxylin A for 24 h or 48 h, co-treated with LPS. The cell proliferation capacity was assessed using cell counting kit-8 (CCK-8), and the cell apoptosis was evaluated by flow cytometry. The Ki67 expression was measured using immunofluorescence and NO production was detected by enzyme linked immunosorbent assay (ELISA) respectively. Western blot analyses were used to investigate the level of cell proliferation related proteins (PCNA, CDK2 and p21) as well as NF-κB, I-κBα and downstream molecules iNOS, IL-6 and TNF-α. The results demonstrated that oroxylin A increased cell survival of LPS treated hPDLSCs in a dose-dependent and time-dependent manner. In addition, oroxylin A treatment inhibited cell apoptosis in hPDLSCs. Furthermore, the levels of NO, NF-κB, iNOS, IL-6 and TNF-α were significantly reduced. And the expression of Ki67, I-κBα, PCNA and CDK2 were significantly increased. Taken together, these findings indicate that oroxylin A promote proliferation and suppress apoptosis in a dose-dependent and time-dependent manner. Oroxylin A may affects LPS induced biological activity via inhibiting NF-κB activation and proinflammatory cytokines expression in hPDLSCs.

Aim: With the widespread use of SGLT2i, various adverse events (AEs) have been reported. This study aimed to describe the distribution of SGLT2i-related AEs in different systems, quantify the association of important medical events (IMEs) and SGLT2i regimens, and build a signal profile of SGLT2i- induced IMEs. Methods: Data from 2015 Q1 to 2020 Q4 in the FDA Adverse Event Reporting System database (FAERS) were selected to conduct disproportionality analysis. Two signal indicators, the reported odds ratio (ROR) and information component (IC), were used to evaluate the correlation between SGLT2i and IMEs. The lower end of the 95% confidence interval of IC (IC025) exceeding zero was deemed a signal. For ROR, it was defined a signal if ROR025 over one, with at least 3 cases. Results: A total of 45,771,436 records were involved, including 111,564 records related to SGLT2i, with 38,366 records of SGLT2i-induced IMEs. Overall, SGLT2i was significantly associated with IMEs (IC=0.36, 95% CI: 0.35-0.38; ROR=1.44, 95% CI: 1.42-1.46). Most SGLT2i-related adverse events occurred in monotherapy (92.93%). Diabetic ketoacidosis was the most IMEs. Specifically, acute osteomyelitis has the strongest signal of all SGLT2i (IC025=7.83), and it was unique to canagliflozin. Diabetic ketoacidosis, acute kidney injury, ketoacidosis, Fournier’s gangrene, and euglycemic diabetic ketoacidosis were common to the four FDA-approved SGLT2i. Conclusion: Our study demonstrated that different SGLT2i regimens lead to different important adverse events, but there are overlapping events. Early identification and management of SGLT2i-associated IMEs are essential for clinical practice.

Microcapsules containing soybean oil were developed with kafirin and sodium caseinate as wall materials by spray drying and characterized by gas chromatographic (GC), Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), physical properties and oxidative stability. The results showed that the encapsulation efficiency of microcapsules was 40.15% under the optimal conditions. Less than 3% loss of unsaturated fatty acids was observed after encapsulation. Particles were spherical, most of the particle surfaces were concave and shriveled, and there were almost no cracks or fissures. In addition, the microencapsulated soybean oil showed lower oxidation values compared to unencapsulated oil, highlighting a protective effect against free oil. In conclusion, kafirin and sodium caseinate as wall materials can potentially be applied to the microencapsulation of vegetable oil for food applications.

Background Despite titanium (Ti) implants have been commonly used in the medical device field due to their superior biocompatibility, implant-associated bacterial infection remains a major clinical complication. Nanosilver, an effective antibacterial agent against a wide spectrum of bacterial strains, with a low-resistance potential, has attracted much interest too. Incorporation of nanosilver on Ti implants may be a promising approach to prevent biofilm formation. Purpose The objective of the study was to investigate the antibacterial effects and osteoinductive properties of nanosilver/poly (dl-lactic-co-glycolic acid)-coated titanium (NSPTi). Methods Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) and the Gram-negative opportunistic pathogen Pseudomonas aeruginosa (PAO-1) were used to evaluate the antibacterial activity of NSPTi implants through the analysis of bacterial colonization in vitro and in vivo. Furthermore, we examined the osteoinductive potential of NSPTi implants by investigating the proliferation and differentiation of MC3T3-E1 preosteoblast cells. In vivo, the osteoinductive properties of NSPTi implants were assessed by radiographic evaluation, H&E staining, and Masson's trichrome staining. Results In vitro, bacterial adhesion to the 2% NSPTi was significantly inhibited and

ZnO nanorod arrays (NRAs) are successfully decorated with Ag2CO3 nanoparticles via the successive ionic layer adsorption and reaction (SILAR) technique. The content of Ag2CO3 is controlled by changing the times of SILAR. The average diameter of ZnO is about 200 nm, and the sizes of Ag2CO3 nanoparticles vary from 50 to 100 nm. Comparison with pure ZnO NRAs, ZnO/Ag2CO3 NRAs show an enhancement of absorption in the visible region, and the absorption intensity in the visible region enhances with the increasing of Ag2CO3 content. The photocatalytic result shows that ZnO/Ag2CO3 NRAs exhibit superior photocatalytic activity than pure ZnO in degrading of rhodamine B (RhB) water solution under simulated sunlight illumination. When the molar ratio of Ag/Zn is about 3.65%, ZnO/Ag2CO3 NRAs exhibit the highest photocatalytic activity.

Nanoassemblies for bioimaging in living cells have attracted much attention. In this work, an efficient and sensitive turn-on fluorescent detection platform for reactive oxygen species (ROS) in living cells has been constructed using the nanoassemblies of upconversion nanoparticles (UCNPs)-MoS2 nanoflakes. In this nanostructure, the positively charged UCNPs could assemble with negatively charged poly(acrylic acid) modified MoS2 nanoflakes by electrostatic force, which led to the remarkable fluorescence quenching of UCNPs owing to the excellent quenching ability of MoS2 nanoflakes. In contrast, in the presence of ROS, the fluorescence recovery of UCNPs is successfully realized owing to the decomposition of MoS2 nanoflakes through the chemical reaction with ROS. The ROS-mediated disassembly of the nanoarchitecture display excellent fluorescent response toward ROS. Furthermore, the disassembly of UCNPs-MoS2 nanoflakes has been successfully used for ROS bioimaging in cells and zebrafish.

Background Biomechanical forces are vital for the regulation of skeletal tissue. Mechanical stretch plays a vital role in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) during orthodontic treatment. Cyclic mechanical stretch may trigger the up-regulated production of reactive oxygen species (ROS). ROS has a critical effect on bone cell function and the pathophysiology of bone diseases. N-acetylcysteine (NAC), a ROS scavenger, possesses powerful antioxidant capacity. The aim of this study was to determine the role of ROS and NAC in PDLSCs during osteogenic differentiation under cyclic mechanical stretch. We further investigated that the therapeutic potential of NAC to improve the changes of the microstructure of alveolar bone during orthodontic tooth movement in rats by micro-CT system. Methods The expression of COL1 (collagen type I), RUNX2 (runt-related transcription factor 2) and OPN (osteopontin) by qRT-PCR and Western blot experiments, and alkaline phosphatase (ALP) staining as well as ALP activity tests were used to examine osteogenic differentiation tendency of PDLSCs subjected to cyclic mechanical stretch of 10% and 0.5Hz deformation induced by the Flexcell tension system. ROS production in PDLSCs were measured under cyclic mechanical stretch by Flow Cytometry. The levels of reduced glutathione (GSH), oxidized GSH (GSSG) and the GSH/GSSG ratio with or without NAC treatment were analyzed. And we evaluated the changes of the microstructure of alveolar bone during orthodontic tooth movement in rats employing micro-CT system. Results NAC treatment could promote the osteogenic differentiation of PDLSCs under cyclic mechanical stretch. Down-regulated ROS generation and the up-regulated level of GSH and the ratio of GSH/GSSG in PDLSCs treated with NAC were observed in response to cyclic mechanical stretch. NAC improved the microstructure of alveolar bone, including BV/TV (bone volume/total volume), Tb.Th (trabecular thickness), Tb.Sp (trabecular separation) and SMI (microstructure model index), during orthodontic tooth movement in rats. Conclusion These results revealed that NAC might be a potential therapeutic approach for the remodeling of the alveolar bone during orthodontic tooth movement.

Bone regeneration is the ultimate goal of periodontal therapies, in which osteogenic differentiation of human periodontal ligament stem cells plays a critical role. The tripartite motif (TRIM)16, an E3 ubiquitin ligase, is downregulated in periodontal tissues of patients with periodontitis, while the role of TRIM16 in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is largely unknown. Firstly, we found that TRIM16 was increased throughout the osteogenic media induced differentiation of hPDLSCs. Then overexpression plasmids and specific short-hairpin RNAs (shRNAs) were constructed to manipulate the expression of target molecules. TRIM16 significantly promoted alkaline phosphatase activity, mineralized nodule formation, and positively regulated the expression of osteo-specific markers RUNX2, COL1A1 and OCN except the mRNA of RUNX2. Mechanistically, TRIM16 serves as a pivotal factor that stabilizes RUNX2 protein levels by decreasing CHIP-mediated K48-linked ubiquitination degradation of the RUNX2 protein. This study identified a novel mechanism of TRIM16 in regulating stability of the RUNX2 protein, which promoted the osteogenic differentiation of hPDLSCs. TRIM16 may be a potential target of stem cell based-bone regeneration for periodontal therapies.

Mild-temperature (

Zwitterions with super-hydrophilic feature are promising membrane materials for water-selective permeation process. However, the applications of zwitterionic materials in membrane are mainly restricted to surface modification or acting as minor additive, while the construction of zwitterionic membrane matrix remains challenging. Herein, we proposed a facile approach to constructing polyzwitterion membrane via direct assembly of graphene oxide (GO)-based core-brush nanosheets. The in situ free radical polymerization of sulfobetaine methacrylate (SBMA) monomer on GO surface ensures the ultra-high grafting amount of poly(sulfobetaine methacrylate) (PSBMA) brushes and then results in the polymer-dominated structure of PSBMA@GO membrane (polymer content high to 83%) instead of the ordered laminar structure in common GO-based membranes. Meanwhile, the original functional groups on GO can be retained after grafting PSBMA, which act as reactive sites for subsequent cross-linking and structural manipulation. Because of the exceptionally high hydration ability and water affinity of PSBMA brushes from the high-density ionic groups, and the densified membrane structure through incorporating the optimized amount of ethylenediamine (EDA) cross-linker, the as-prepared polyzwitterion membrane obtains an excellent water/ethanol separation performance with the separation factor of 1420 and the permeate flux of 5.20 kg/m2·h (70 oC, 20 wt% water in feed), which shows remarkable superiority compared with the reported polymeric membranes. The strategy proposed here provides a novel pathway for constructing polyzwitterion membranes with high separation efficiency, and can be applied with a greater scope of monomers.

Periodontitis is a chronic inflammatory disease that result in severe loss of supporting structures and substantial tooth loss. Oxidative stress is tightly involved in the progression of periodontitis. Tripartite Motif 16 (TRIM16) has been identified as a novel regulatory protein in response to oxidative and proteotoxic stresses. The present study aimed to investigate the role of TRIM16 in human periodontal ligament stem cells (hPDLSCs) under oxidative stress. First, we found that the expression of TRIM16 decreased after exposure to H2O2. Then TRIM16 overexpression alleviated H2O2-induced oxidative stress by enhancing antioxidant capacity and reducing the amount of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS). TRIM16 increased cell viability, inhibited cell apoptosis and the depolarization of the mitochondrial membrane potential in hPDLSCs. Furthermore, TRIM16 attenuated H2O2-induced suppression of osteogenic differentiation. Mechanistically, TRIM16 promoted the activation of protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT), p-Akt and Nrf2, while knockdown of PICOT reversed TRIM16-mediated ROS resistance and decreased the expression of p-Akt and Nrf2. In conclusion, TRIM16 alleviated oxidative damage in hPDLSCs via the activation of PICOT/Akt/Nrf2 pathway, suggesting that TRIM16 could be a promising target to develop effective therapies for periodontitis.

A sandwich-structured SnS2@reduced graphene oxide (SnS2@rGO) nanocomposite consisting of SnS2 sheets uniformly anchored on both sides of rGO sheets is presented. The SnS2@rGO nanocomposites, prepared through a dipping process combining with a hydrothermal method, show good electrochemical performance, including the high reversible capability of 959.2 mA h g−1 at 500 mA g−1 over 500 cycles, and superior rate performance. A full cell consisting of a commercial LiCoO2 cathode and the SnS2@rGO anode delivers the stable capacity of ∼821.3 mA h g−1 (anode basis) after 200 cycles at 100 mA g−1. The sandwich-structured SnS2@rGO nanocomposite accommodates volume changes during Li+ insertion and extraction and facilitates the rapid transport of ions and electrons. This high performance indicates that the presented SnS2@rGO nanocomposite is a promising anode candidate for practical application in Li-ion batteries.

ZnO/Ag2S core–shell nanorod arrays (NRAs) are successfully fabricated via chemical bath deposition procedure and subsequent successive ionic layer adsorption and reaction (SILAR) technique. The average diameter of ZnO core is about 100 nm, and the thicknesses of Ag2S shell are adjusted from 6 to 35 nm by varying the time of SILAR. ZnO/Ag2S NRAs show an enhancement of absorption in the visible region, and the absorption intensity in the visible region enhances with the increase in Ag2S shell thickness. Compared with pure ZnO NRAs, ZnO/Ag2S NRAs exhibit outstanding photocatalytic activity for degradation of rhodamine B (RhB) aqueous solution under simulated sunlight irradiation. In addition, ZnO/Ag2S NRAs with Ag2S shell thickness of 6 nm display the highest photocatalytic activity, which is 6.0 times higher than that of pure ZnO NRAs.

A general method for preparing sandwiched nanostructures consisting of reduced graphene oxide (rGO) and metal sulfides is presented. As a demonstrating example, the sandwiched MoS2@rGO nanocomposites show a good electrochemical performance including a high reversible capability of 1345 mAh g−1 at 0.5 A g−1 over 400 cycles, and a superior rate performance. Moreover, this method has been verified to fabricate a large set of transition metal sulfides- and double metal sulfides-based rGO composites such as CoS@rGO, NiS@rGO, NiCo2S4@rGO, and CuCo2S4@rGO, enabling the preparing approach presented here applicable for many possible anode and cathode chemistries. Impact statement A general strategy for the fabrication of sandwiched nanostructures consisting of reduced graphene oxide (rGO) and metal sulfides for highly reversible Li-ion battery anode has been developed.

Porous ZnS/ZnSe nanosheets (NSs) with different ratios of the two components are fabricated by a facile two-step solvothermal method with the comparison to ZnS and ZnSe NSs. ZnS/ZnSe NSs have the similar geometrical morphology with the pure ZnS and ZnSe NSs, and show a better absorption in the UV light region as well as in the visible-light region. Compared with pure ZnS or ZnSe, ZnS/ZnSe NSs display an outstanding photodegradation ability for decolorization of methyl orange (MO) under visible light irradiation. ZnS/ZnSe NSs with a molar ratio of ZnS to ZnSe as 1:1 exhibit the highest photocatalytic activity and a good photocatalytic stability, and their degradation rate constants are nearly 53 and 6 times higher than those of pure ZnS and ZnSe NSs.

Nano-engineering graphene-based composite anodes have motivated broad investigations of the high energy density lithium-ion batteries. Here, a sandwich-like and porous NiCo2O4@reduced graphene oxide (rGO) nanocomposite was presented. Through an incipient dipping process, the NiCo2O4 precursors attach on the rGO densely, which significantly improves the material loading of the NiCo2O4@rGO composites. Serving as an anode of the lithium-ion battery, the NiCo2O4@rGO composites exhibit an unprecedented capacity of 1566 mAh g−1 cycling at a current density of 1000 mA g−1 over 700 times; while a capacity of 886 mAh g−1 at 100 mA g−1 over 100 cycles was achieved upon a NiCo2O4@rGO//LiCoO2 full-cell, which indicates a promising potential for high energy density battery applications.

During orthodontic treatment, mechanical stretch serves a crucial function in osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Up-regulated reactive oxygen species (ROS) level is a result of cyclic mechanical stretch in many cell types. Nuclear factor erythroid-2-related factor-2 (Nrf2) is a master regulator in various antioxidants expression. However, it is not known whether cyclic mechanical stretch could induce the ROS generation in PDLSCs and whether Nrf2 participated in this process. The present study was aimed to investigate the role of Nrf2 in PDLSCs under cyclic mechanical stretch. Our results showed that cyclic mechanical stretch increased ROS level and the nuclear accumulation of Nrf2 during osteoblast differentiation. Knocking down Nrf2 by siRNA transfection increased ROS formation and suppressed osteogenic differentiation in PDLSCs. T-BHQ, a Nrf2 activator, promoted the osteogenic differentiation in PDLSCs under cyclic mechanical stretch, and improved the microstructure of alveolar bone during orthodontic tooth movement in rats by employing micro-CT system. Taken together, Nrf2 activation was involved in osteogenic differentiation under cyclic mechanical stretch in PDLSCs. T-BHQ could promote the osteogenic differentiation in vitro and in vivo, suggesting a promising option for the remodeling of the alveolar bone during orthodontic tooth movement.

Total organic carbon (TOC) content prediction is an important index for evaluating geochemical characteristics of source rocks. Although the methods based on multiple regression and shale content index classification regression can predict TOC content of mudstone in different layers, the models are relatively simple and the results are not accurate. In this paper, the prediction method of artificial neural network is used to predict TOC content of Neogene source rocks in Liping area, Qaidam Basin. The results show that: (1) the correlation between TOC prediction curve and parameters of logging curve is significant; (2) the TOC prediction curve has obvious fluctuation, reflecting the strong heterogeneity of the stratigraphic organic matter; (3) the predicted TOC value is closer to the measured value. Using this method to predict TOC content can be widely used for oil and gas exploration and development in future.

Cyclical stretch-induced bone formation during orthodontic treatment is a complex biological process modulated by various factors including miRNAs and their targeted-gene network. However, the miRNA expression profile and their roles in osteogenic differentiation of bone mesenchymal stem cells (BMSCs) exposed to mechanical stretch remains unclear. Here, we use the miRNA microarray assay to screen for mechano-sensitive miRNAs during stretch-induced osteogenic differentiation of BMSCs and identified that nine miRNAs were differentially expressed between stretched and control BMSCs. Furthermore, miR-503-5p, which was markedly downregulated in both microarray assay and qRT-PCR assay were selected for further functional verification. We found that overexpression of miR-503-5p in BMSCs attenuated stretch-induced osteogenic differentiation while the effect was reversed by miR-503-5p inhibition treatment. In vivo studies, overexpression of miR-503-5p with specific agomir decreased Runx2, ALP mRNA, and protein expression, decreased osteoblast numbers and osteoblastic bone formation in the OTM tension sides. In conclusion, our study revealed that miR-503-5p functions as the mechano-sensitive miRNA and inhibits BMSCs osteogenic differentiation subjected to mechanical stretch and bone formation in OTM tension sides.

It is crucial to investigate the evolution of microstructure to understand the deterioration mechanisms of shotcrete under cyclic freezing and thawing (F-T). In this study, nanoindentation and X-ray micro-CT techniques are integrated to characterize microstructural damage of shotcrete and its evolution due to F-T actions. An integrated statistical method of nanoindentation and micro-CT (NI-MCT) is developed with the Gaussian mixture deconvolution method to demonstrate the increase and correlation of the heterogeneous phase in cement paste and interfacial transition zones in the process of F-T cycles. The micromechanical property allows to segment the micro-CT images of cement paste into pores/cracks, porous phase, calcium silicate hydrate, and calcium hydroxide/unhydrated clinker. The integrated NI-MCT method can provide new knowledge on the evolution law of F-T-induced damage in cement paste and serve as an effective tool in characterizing and quantifying the damage and its evolution process for cement-based heterogeneous materials.

Due to extensive metastasis, poor blood supply, and drug-resistant, there is still no effective clinical means to treat peritoneal dissemination of gastric cancer. Here, an aptamer-siRNA chimera (Chim)/polyethyleneimine (PEI)/5-fluorouracil (5-FU)/carbon nanotube (CNT)/collagen membrane is constructed, which could be divided into 15 layers with a thickness of 70-100 µm. Sustained release experiments show that the collagen membranes can control 5-FU release for more than 2 weeks. Aptamer-siRNA chimera can specifically bind to gastric cancer cells, enabling targeted delivery of 5-FU and silencing drug-resistant gene. In vitro experiments demonstrated that Chim/PEI/5-FU/CNT nanoparticles promoted the apoptosis of 5-FU-resistant gastric cancer cells, inhibited their invasion and proliferation. Animal experiments show that Chim/PEI/5-FU/CNT/collagen membrane significantly inhibits the expression of mitogen-activated protein kinase (MAPK), and effectively treats peritoneal dissemination of 5-FU-resistant gastric cancer. Compared with siRNA/PEI/5-FU/CNT group, ki-67 proliferation index, and matrix metallopeptidase 9 (MMP9) expression are significantly decreased in the Chim/PEI/5-FU/CNT group, while the proportion of apoptotic cells is markly increased. In conclusion, a chimera/PEI/5-FU/CNT/collagen membrane is constructed, which can effectively treat peritoneal dissemination of drug-resistant gastric cancer. The study provides a new therapeutic approach for relevant clinical treatment.

Keqiang Huang, Dongxu Liu Department of Orthodontics, School of Stomatology, Shandong University, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan 250012, China Background: Erlotinib is a commonly used molecular-targeted drug for the treatment of tongue cancer. However, the development of acquired resistance to erlotinib hampers its therapeutic use. Materials and methods: To analyze the erlotinib resistance, long-term and short term survival assay were used to compare the resistance between parental and resistant tongue cancer cells. Flow cytometry, Hochest staining and western blot were used to analyze the apoptosis among the cells. Moreover, Transwell and wound healing assay were used to compare the invasion ability of the cells. To deeply explore the drug resistance in vivo, orthotopic tumor studies were applied. Finally, to explain the mechanism of c-met in erlotinib resistance, shRNA against c-met was used to down-regulate the expression of c-met. And SU11274 also used in orthotopic model. Results: We established erlotinib-resistant human tongue cancer cell line by chronic exposure of TCA-8113 cells to increasing concentrations of erlotinib and determined the role of c-MET and EGFR in the development of acquired resistance. We found a significant increase in the phosphorylation of c-MET and an obvious decrease of the phosphorylation of EGFR in erlotinib-resistant cells. Our results also revealed that inhibition of c-MET alone with SU11274 exerted an inhibitory effect on the proliferation of erlotinib-resistant cells in the short term; however, it failed to sustain the inhibitory effect in the long term. Simultaneous inhibition of c-MET and EGFR significantly inhibited the proliferation of erlotinib-resistant cells in both a short and long period. Furthermore, we explored the underlying mechanism and found that treatment of erlotinib-resistant cells with SU11274 or shRNA against c-MET induced the phosphorylation of EGFR. Moreover, our results demonstrated that simultaneous inhibition of c-MET and EGFR significantly inhibited the migration and invasion of erlotinib-resistant cells. Conclusion: Taken together, our results suggested that c-MET is involved in acquired drug resistance to erlotinib and that cotargeting of EGFR and c-MET could overcome acquired resistance to erlotinib and inhibit the invasion and metastasis of erlotinib-resistant cells. Keywords: tongue cancer, erlotinib, acquired resistance, EGFR, c-MET

Objective This study aims to discuss long noncoding RNA (lncRNA) maternally expressed gene 3 (MEG3) function of regulating osteogenesis in human periodontal ligament cells (hPDLCs). Methods First, use of a mineralizing solution induced osteogenic differentiation of hPDLCs to establish a differentiated cell model. Through microarray analysis, we selected a lncRNA MEG3 with marked changes between differentiated and undifferentiated cells. The quantitative polymerase chain reaction was used to detect the MEG3 content and an enzyme-linked immunosorbent assay was used to detect changes in related proteins. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and apoptosis was measured by flow cytometry. Alizarin red staining was also used to evaluate cells' osteogenic level. Finally, RNA-binding protein immunoprecipitation assays were conducted to further clarify the endogenous relationship between MEG3 and bone morphogenetic protein 2 ( BMP2) in hPDLCs. Results MEG3 was downregulated in osteogenic differentiation hPDLCs induced by mineralizing solution. Overexpression of MEG3 inhibited cell viability and increased cell apoptosis. MEG3 overexpression can reverse osteogenic differentiation induced by mineralizing solution. MEG3 can suppress BMP2 through interaction with heterogeneous nuclear ribonucleoprotein I. Conclusion Upregulation of MEG3 inhibits the osteogenic differentiation of periodontal ligament cells by downregulating BMP2 expression.

Background/Aims: In this study, we aimed to use bioinformatics tools to identify the specific miRNAs and mRNAs involved in osteogenic differentiation and to further explore the way in which miRNA regulates osteogenic differentiation. Methods: The microarray GSE80614, which includes data from 3 human mesenchymal stromal cells (hMSCs) and 3 hMSCs after 72 hours (hr) of osteogenic differentiation, was used to screen for differentially expressed mRNAs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of these mRNAs were conducted using Gene Set Enrichment Analysis (GSEA). Then, the miRanda website was employed to detect the binding sites of DHRS3. In vitro experiments, including RT-PCR and western blotting, were used to detect miR-233 and DHRS3 expression levels 7 and 14 days (d) after the induction of osteogenic differentiation using human bone marrow-derived mesenchymal stem cells (hBMSCs). The target relationship between miR-223 and DHRS3 was confirmed by a dual luciferase assay. ALP (alkaline phosphatase) staining, ARS (Alizarin Red S) staining and western blotting (Runx2, OPN, OCN) were used to detect the level of osteogenic differentiation after transfection with miR-223 mimics and DHRS3 cDNA. Results: In this study, 127 mRNAs differentially expressed during osteogenic differentiation were identified in GSE80614. GO term and KEGG pathway enrichment analyses found that the retinol metabolism pathway was activated during osteogenic differentiation and that DHRS3, which is involved in the pathway, was upregulated. During osteogenic differentiation in hBMSCs, miR-223 was gradually downregulated, while DHRS3 was upregulated. After 14 days of osteogenic differentiation, ALP and ARS staining assay results showed strong ALP activity and extracellular matrix calcification with the inhibition of miR-223 or the overexpression of DHRS3. Furthermore, the expression levels of Runx2, OPN, and OCN were upregulated with the knockdown of miR-223 or the overexpression of DHRS3, while the simultaneous transfection of a miR-223 agomir and DHRS3 cDNA resulted in no significant difference from the negative control (NC) group. Conclusion: The inhibition of miR-223 promotes the osteogenic differentiation of hBMSCs via the upregulation of DHRS3.

High purity stoichiometric lithium niobate nanocrytalline were synthesized with the low-temperature soft-chemical route. All the products were characterized with X-ray powder diffraction (XRD), scanning electron microscopy (SEM) and high resolution transition electron microscopy (HRTEM). An outstanding advantage of such a soft-chemical synthesis route is that the reaction temperature required to produce niobate crystalline is much lower than those in other methods. Structure characterization results indicated that the LiNbO 3 powders prepared had rhombohedral morphology. X-ray diffraction and HRTEM results of the as-prepared sample indicate that the sample is well crystallized. The Raman and Fourier transform (FT) infrared properties of the niobates were studied.

Graphene aerogels have many advantages, such as low density, high elasticity and strong adsorption. They are considered to be widely applicable in many fields. At present, the most valuable research area aims to find a convenient and effective way to prepare graphene aerogels with excellent properties. In this work graphene/carbon nanotube aerogels are prepared through hydrothermal reduction, freeze-drying and high temperature heat treatment with the blending of graphene oxide and carbon nanotubes. A new reducing agent-ascorbic acid is selected to explore the best preparation process. The prepared aerogels have compression and resilience and oil absorption properties due to the addition of carbon nanotubes as designed.

p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-kappaB in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-kappaB activation during myogenesis, not through down-regulation of degradation of IkappaB-alpha, and consequent translocation of the p65 subunit of NF-kappaB to the nucleus. It is likely that stretch-induced NF-kappaB activation by phosphorylation of p65 NF-kappaB. Moreover, depletion of p38alpha using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-kappaB activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-kappaB signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The alpha isoform of p38MAP kinase regulates the transcriptional activation of NF-kappaB following stimulation with cyclic stretch.

Potassium nitrate catalysts supported on different oxides (CeO 2 , Ce 0.5 Zr 0.5 O 2 and ZrO 2 ) were prepared for diesel soot combustion. The ageing treatment was performed at 800 °C for 24 h and the catalytic activity was evaluated by a temperature-programmed oxidation technique. The results demonstrated that, compared with CeO 2 and ZrO 2 , Ce 0.5 Zr 0.5 O 2 presented good redox properties, a high surface area and available potassium-holding capacity at an elevated temperature. For aged K/Ce 0.5 Zr 0.5 O 2 , the combustion temperature of soot particle was 359 °C under tight contact conditions and 455 °C under loose contact conditions. Thus, ceria–zirconia mixed oxides were considered as good candidate supports for diesel soot oxidation catalysis.

In this study, a simple, efficient enzymatic production process for the environmentally friendly synthesis of l-citrulline from l-arginine was developed using arginine deiminase (ADI) from Lactococcus lactis. Following overexpression of L. lactis ADI in Escherichia. coli BL21 (DE3) and experimental evolution using error-prone PCR, mutant FMME106 was obtained with a Km for l-arginine of 3.5mM and a specific activity of 195.7U/mg. This mutant exhibited a maximal conversion of 92.6% and achieved a final l-citrulline concentration of 176.9g/L under optimal conditions (190g/L l-arginine, 15g/L whole-cell biocatalyst treated with 2% isopropanol for 30min, 50°C, pH 7.2, 8h). The average l-citrulline synthesis rate of 22.1g/L/h is considerably higher than that reported for other similar biocatalytic approaches, therefore the process developed in the present work has great potential for large-scale production of l-citrulline.

Orthodontic force may lead to cell damage, circulatory disturbances, and vascular changes of the dental pulp, which make a hypoxic environment in pulp. In order to maintain the homeostasis of dental pulp, hypoxia will inevitably induce the defensive reaction. However, this is a complex process and is regulated by numerous factors. In this study, we established an experimental animal model of orthodontic tooth movement to investigate the effects of mechanical force on the expression of VEGF and HIF-1αin dental pulp. Histological analysis of dental pulp and expressions of HIF-1αand VEGF proteins in dental pulp were examined. The results showed that inflammation and vascular changes happened in dental pulp tissue in different periods. Additionally, there were significant changes in the expression of HIF-1αand VEGF proteins under orthodontic force. After application of mechanical load, expression of HIF-1αand VEGF was markedly positive in 1, 3, 7 d, and 2 w groups, and then it weakened in 4 w group. These findings suggested that the expression of HIF-1αand VEGF was enhanced by mechanical force. HIF-1αand VEGF may play an important role in retaining the homeostasis of dental pulp during orthodontic tooth movement.

Gram-negative bacterial endotoxemia may lead to the pathological increase of vascular permeability with systemic vascular collapse, a vascular leak syndrome, multiple organ failure (MOF), and/or shock. Previous studies demonstrated that C1 inhibitor (C1INH) protects mice from lipopolysaccharide (LPS)–induced lethal septic shock via a direct interaction with LPS. Here, we report that C1INH blocked the LPS-induced increase in transendothelial flux through an endothelial monolayer. In addition, LPS-mediated detachment of cultured endothelial cells was prevented with C1INH. C1INH also inhibited LPS-induced endothelial cell apoptosis as demonstrated by suppression of DNA fragmentation and annexin V expression. As illustrated by laser scanning confocal microscopy, C1INH completely blocked the binding of fluorescein isothiocyanate (FITC)–LPS to human umbilical vein endothelial cells (HUVECs). C1INH protected from localized LPS-induced increased plasma leakage in C57BL/6J mice and in C1INH-deficient mice. Local vascular permeability in response to LPS was increased to a greater extent in C1INH-deficient mice compared with wild-type littermate controls and was reversed by treatment with C1INH. Systemic administration of LPS to mice resulted in increased vascular permeability, which was reduced by C1INH. Therefore, these studies demonstrate that C1INH, in addition to its role in suppression of LPS-mediated macrophage activation, may play an important role in the prevention of LPS-mediated increased vascular permeability, endothelial cell injury, and multiple organ failure.

C1 inhibitor (C1INH) is beneficial in animal models of endotoxemia and sepsis. However, the mechanism(s) of C1INH protection remain(s) ill-defined. In this study, we demonstrated that both active C1INH and reactive center-cleaved, inactive C1INH protected mice from lethal Gram-negative endotoxemia. Both forms of C1INH blocked the LPS-binding protein-dependent binding of Salmonella typhimurium LPS to the murine macrophage cell line, RAW 264.7, and suppressed LPS-induced TNF-α mRNA expression. Inhibition of LPS binding to RAW 264.7 cells was reversed with anti-C1INH Ab and was more efficient when C1INH was incubated first with LPS rather than with the cells. C1INH also suppressed LPS-induced up-regulation of TNF-α mRNA in whole human blood. The interaction of C1INH with LPS was directly demonstrated both by ELISA and by nondenaturing PAGE, but deletion of the amino-terminal 97-aa residues abrogated this binding. Therefore, C1INH, in addition to its function as a serine protease inhibitor, has a novel anti-inflammatory function mediated via its heavily glycosylated amino-terminal non-serpin domain.

IFN-gamma induction of C1 inhibitor (C1INH) is mediated by an IFN-gamma-activated sequence (GAS), via binding of signal transducer and activator of transcription 1 (STAT1). These studies focused on the factors responsible for down-regulation of nuclear STAT1 in hepatocytes, the primary site of synthesis of C1INH. The activity of nuclear STAT1 following stimulation with IFN-gamma was sustained with the phosphatase inhibitor, pervanadate, or the proteasome inhibitor, lactacystin. Pervanadate prolonged STAT1 activation and blocked the inactivation of nuclear STAT1. Binding of ubiquitin to phosphorylated STAT1 was detectable in cells treated with lactacystin. Staurosporine only moderately decreased the prolongation of nuclear phosphorylated STAT1 after pretreatment with pervanadate or lactacystin. An antisense mitogen-activated protein kinase phosphatase (MKP-1) oligonucleotide prolonged the accumulation of phosphorylated STAT1. These data are consistent with the hypothesis that down-regulation of IFN-gamma-mediated nuclear STAT1 binding in hepatocytes involves both dephosphorylation by MKP-1 and degradation via proteolysis by the ubiquitin-dependent proteasome pathway.

Adipose tissue macrophages (ATMs) undergo a phenotypic switch from alternatively activated antiinflammatory M2 macrophages in lean individuals to classically activated proinflammatory M1 macrophages in obese subjects. However, the molecular mechanism underlying this process remains unclear. In this study we aim to determine whether DNA methyltransferase 3b (DNMT3b) regulates macrophage polarization and inflammation. We found that the expression of DNMT3b was significantly induced in macrophages exposed to the saturated fatty acid stearate, was higher in ATMs isolated from obese mice, but was significantly lower in alternatively activated M2 vs classically activated M1 ATMs, suggesting a role for DNMT3b in regulation of macrophage polarization and inflammation in obesity. DNMT3b knockdown promoted macrophage polarization to alternatively activated M2 phenotype and suppressed macrophage inflammation, whereas overexpressing DNMT3b did the opposite. Importantly, in a macrophage-adipocyte coculture system, we found that DNMT3b knockdown significantly improved adipocyte insulin signaling. The promoter of peroxisome proliferator activated receptor (PPAR)γ1, a key transcriptional factor that regulates macrophage polarization, is enriched with CpG sites. Chromatin immunoprecipitation assays showed that DNMT3b bound to the methylation region at PPARγ1 promoter, which was further enhanced by stearate. Moreover, pyrosequencing analysis revealed that stearate increased DNA methylation at PPARγ1, which was prevented by DNMT3b deficiency. Therefore, our data demonstrate that DNMT3b plays an important role in regulating macrophage polarization through epigenetic mechanisms. In obesity, elevated saturated fatty acids enhance DNMT3b expression, leading to DNA methylation at the PPARγ1 promoter, which may contribute to deregulated adipose tissue macrophage polarization, inflammation, and insulin resistance.

Three polyurethane films were prepared by adding the antioxidant-1010 and the composite stabilizer to the polyurethane matrix, respectively. The accelerated weathering tests were performed by using self-designed UV/ozone aging test device. The color difference, yellowness index, UV-Vis spectrum, and infrared spectrum were recorded with colorimeter apparatus, UV-Vis spectroscopy, and FT-IR spectroscopy, respectively. The results show that, for the polyurethane film, the composite stabilizer can remarkably decrease UV transmission, the antioxidant-1010 and the composite stabilizer can markedly decrease the photooxidation index and the carbonyl index, respectively, and the antioxidant-1010 can significantly improve the antiyellowing properties after 60 h exposure. With incremental exposure time for the three films, UV-Vis transmission decreases, the photooxidation index, the carbonyl index, color difference, and yellowness index increase gradually. Under current experimental conditions, the order of UV/O3aging resistance from highness to lowness is as follows: the polyurethane film modified by the antioxidant-1010, the polyurethane film modified by composite stabilizer, and the pure polyurethane film.

The pure polyester polyurethane (TPU) film and the modified TPU (M-TPU) film containing 2.0 wt.% inorganic UV absorbers mixture (nano-ZnO/CeO2with weight ratio of 3 : 2) and 0.5 wt.% organic UV absorbers mixture (UV-531/UV-327 with weight ratio of 1 : 1) were prepared by spin-coating technique. The accelerated aging tests of the films exposed to constant UV radiation of 400 ± 20 µW/cm2(313 nm) with an ozone atmosphere of 100 ± 2 ppm were carried out by using a self-designed aging equipment at ambient temperature and relative humidity of 20%. The aging resistance properties of the films were evaluated by UV-Vis spectra, Fourier transform infrared spectra (FT-IR), photooxidation index, and carbonyl index analysis. The results show that the composite UV absorber has better protection for TPU system, which reduces distinctly the degradation of TPU film. O3/UV aging of the films increases with incremental exposure time. PI and CI of TPU and M-TPU films increase with increasing exposure time, respectively. PI and CI of M-TPU films are much lower than that of TPU film after the same time of exposure, respectively. Distinct synergistic aging effect exists between ozone aging and UV aging when PI and CI are used as evaluation index, respectively. Of course, the formula of these additives needs further improvement for industrial application.

C1 inhibitor (C1INH), a complement regulatory protein, prevents endotoxin shock via a direct interaction of the amino-terminal domain with gram-negative bacterial lipopolysaccharide (LPS). Importantly, the cleaved, inactive C1INH still is an anti-endotoxin effector indicating the anti-endotoxin peptide that generates from the amino-terminal domain of C1INH. In this study, we first identified that a cleaved fragment within the major part of the amino-terminal domain in in vitro proteolytic analysis of C1INH had an ability to bind to LPS. We synthesized several peptides overlapping the C1INH cleaved fragment. Among these synthetic peptides, a 13-mer derivative peptide at position from 18 to 30, named N2((18-30)), exhibited the most powerful anti-endotoxin activity in vitro, enlightening that it was most strong at binding to LPS, inhibiting the interaction of LPS with LPS-binding protein (LBP), blocking LPS binding to CD14(+) cells, and suppressing production of tumor necrosis factor (TNF)-alpha by murine macrophages, RAW 264.7. In the murine endotoxin shock model, the peptide N2((18-30)) protected mice from LPS-induced lethal septic shock by inhibiting macrophage activation. These data indicate that the peptide N2((18-30)) derived from the amino-terminal region of C1INH is anti-endotoxin.

C3 is common to all pathways of complement activation augmenting ischemia/reperfusion (I/R)-induced myocardial injury and cardiac dysfunction. Complement inhibition with the complement regulatory protein, C1 inhibitor (C1INH), obviously exerts cardioprotective effects. Here, we examine whether C1INH regulates C3 activity in the ischemic myocardial tissue. C1INH markedly suppressed C3 mRNA expression and protein synthesis in both a model of I/R-induced rat acute myocardial infarction (AMI) and the cultured rat H9c2 heart myocytes. At least, this regulation was at the transcriptional level in response to oxygen tension. In vitro, C3 deposition on, and binding to, the surface of rat myocardial cells were significantly blocked by C1INH treatment. C1INH could inhibit classical complement-mediated cell lysis via suppressing the biological activity of C3. Therefore, C1INH, in addition to inhibition of the systemic complement activation, prevents myocardial cell injury via a direct inhibitory role in the local myocardial C3 activity.

Complement activation augments myocardial cell injury and apoptosis during ischemia/reperfusion (I/R), whereas complement system inhibition with C1 inhibitor (C1INH), a serine protease inhibitor, exerts markedly cardioprotective effects. Our recent data demonstrate that C1INH prevents vascular endothelial cell apoptosis and a "modified" form of the reactive center loop-cleaved, inactive C1INH (iC1INH) plays an anti-inflammatory role in endotoxin shock. The aim of this study was to determine whether C1INH protects against myocardial cell injury via an anti-apoptotic activity or anti-inflammatory effect. In a rat model of acute myocardial infarction (AMI) induced by I/R, administration of C1INH protected against cardiomyocytic apoptosis via normalization of ratio of the Bcl-2/Bax expression in the myocardial infarct area. C1INH improved parameters of cardiac function and hemodynamics and reduced myocardial infarct size (MIS). In addition, myocardial and blood myeloperoxidase (MPO) activity, a marker of neutrophil infiltration, was decreased by treatment of C1INH. In cultured H9c2 rat cardiomyocytic cells, C1INH blocked hypoxia/reoxygenation-induced apoptosis in the absence of sera associated with inhibition of cytochrome c translocation and suppression of caspase-3 activation. The proportion of Bcl-2/Bax expression induced by hypoxia/reoxygenation was reversed by C1INH. Importantly, iC1INH also revealed these similar effects, indicating that C1INH has a direct anti-apoptotic activity. Therefore, these studies support the hypothesis that C1INH, in addition to inhibition of activation of the complement and contact systems, improves outcome in I/R-mediated myocardial cell injury via an anti-apoptotic activity independent of serine protease inhibitory activity.

The C1 inhibitor (C1INH), a plasma complement regulatory protein, prevents endotoxin shock, at least partially via the direct interaction of its amino-terminal heavily glycosylated nonserpin region with gram-negative bacterial lipopolysaccharide (LPS). To further characterize the potential LPS-binding site(s) within the amino-terminal domain, mutations were introduced into C1INH at the three N-linked glycosylation sites and at the four positively charged amino acid residues. A mutant in which Asn3was replaced with Ala was markedly less effective in its binding to LPS, while substitution of Asn47or Asn59had little effect on binding. The mutation of C1INH at all four positively charged amino acid residues (Arg18, Lys22, Lys30, and Lys55) resulted in near-complete failure to interact with LPS. The C1INH mutants that did not bind to LPS also did not suppress LPS binding or LPS-induced up-regulation of tumor necrosis factor alpha mRNA expression in RAW 264.7 macrophages. In addition, the binding of C1INH mutants to diphosphoryl lipid A was decreased in comparison with that of recombinant wild-type C1INH. Therefore, the interaction of C1INH with gram-negative bacterial LPS is dependent both on the N-linked carbohydrate at Asn3and on the positively charged residues within the amino-terminal domain.