Phylogenetic diversity of eastern Asia-eastern North America disjunct plants is mainly associated with divergence time.

The underlying causes of biodiversity disparities among geographic regions have long been a fundamental theme in ecology and evolution. However, the patterns of phylogenetic diversity (PD) and phylogenetic beta diversity (PBD) of congeners that are disjunctly distributed between eastern Asia-eastern North America (EA-ENA disjuncts) and their associated factors remain unknown. Here we investigated the standardized effect size of PD (SES-PD), PBD, and potentially associated factors in 11 natural mixed forest sites (five in EA and six in ENA) where abundant EA-ENA disjuncts occur. We found that the disjuncts in ENA possessed higher SES-PD than those in EA at the continental scale (1.96 vs -1.12), even though the number of disjunct species in ENA is much lower than in EA (128 vs 263). SES-PD of the EA-ENA disjuncts tended to decrease with increasing latitude in 11 sites. The latitudinal diversity gradient of SES-PD was stronger in EA sites than in ENA sites. Based on the unweighted unique fraction metric (UniFrac) distance and the phylogenetic community dissimilarity, PBD showed that the two northern sites in EA were more similar to the six-site ENA group than to the remaining southern EA sites. Based on the standardized effect size of mean pairwise distances (SES-MPD), nine of eleven studied sites showed a neutral community structure (-1.96 ≤ SES-MPD ≤ 1.96). Both Pearson's r and structural equation modeling suggested that SES-PD of the EA-ENA disjuncts was mostly associated with mean divergence time. Moreover, SES-PD of the EA-ENA disjuncts was positively correlated with temperature-related climatic factors, although negatively correlated with mean diversification rate and community structure. By applying approaches from phylogenetics and community ecology, our work sheds light on historical patterns of the EA-ENA disjunction and paves the way for further research.

Sequential thermal dissolution of two low-rank coals and characterization of their structures by high-performance liquid chromatography/time-of-flight mass spectrometry and gas chromatography/mass spectrometry.

RATIONALE: Gas chromatography/mass spectrometry (GC/MS) and high-performance liquid chromatography/time-of-flight mass spectrometry (HPLC/TOF-MS) were used to separate and reveal the molecular characteristics of organic matter in low-rank coals. METHODS: Six soluble portions (SPs) were obtained by sequential thermal dissolution (TD) of two low-rank coals in the order of cyclohexane, acetone and methanol solvents at 300°C. Organic matter with different molecular characteristics were enriched in eachTD extract, which was further separated and analyzed by GC/MS and HPLC/TOF-MS using an electrospray ionization source in positive mode to obtain a comprehensive understanding of the structural composition of coals. RESULTS: Low polarity compounds like alkanes and arenes have a better solubility in cyclohexane. Phorone has the highest relative abundance in the acetone SPs, and the main compounds detected in the methanol SPs are alcohols and phenols. According to the data from HPLC/TOF-MS, most of the oxygen atoms are in the form of carbonyl and alkoxy groups. The nitrogen-containing compounds in SPs are mainly saturated aliphatic amines and pyridines. The sulfur-containing compounds mainly exist in the form of thioalkanes and thiophenes. CONCLUSIONS: Non-destructive methods were used to obtain soluble matter from coals, and different chromatographic and mass spectrometric techniques were used to separate and analyze the organic matter in coals. Detailed molecular structural information was obtained for the efficient and clean utilization of low-rank coals.

Preparation of layered-porous carbon from coal tar pitch narrow fractions by single-solvent extraction for superior cycling stability electric double layer capacitor application.

Coal tar pitch (CTP) with high carbon content and wide source of raw materials was an excellent precursor for the preparation of porous carbons (PCs). CTP was composed of polycyclic aromatic hydrocarbons (PAHs) with complex molecular size and chemical structure, the separation of CTP into several fractions with relatively narrow molecular weight by solvent extraction was of significance for CTP utilization. In this paper, CTP was treated by single-solvent extraction (carbon disulfide, acetone and ethyl acetate), and the six fractions were used as raw materials to prepare PCs as electrode material for electric double layer capacitor. The fractions were well characterized and the effect of mass distribution of different narrow fractions on structure property and electrochemical performance of the PCs was studied. The PCs prepared by carbon disulfide extract, acetone raffinate and ethyl acetate extract, containing more PAHs, exhibited the excellent specific capacitance performance in comparison with its residual components. The remarkable performance might contribute in the enhanced transport of electrolyte ions via the molecular graphene structure of PAHs. Additionally, the PC prepared by carbon disulfide raffinate showed outstanding cycling performance (99.7% at 2 A g-1 after 15,000 cycles) which was related to its unique layered porous structure.

N/O co-doped porous interconnected carbon nanosheets from the co-hydrothermal treatment of soybean stalk and nickel nitrate for high-performance supercapacitors.

Porous interconnected carbon nanosheets (PICNs) with high electrochemical performance were prepared by doping urea and a co-hydrothermal precursor derived from soybean stalk (SS) and nickel nitrate. The specific surface area and average pore diameter of the as-synthesized PICNs are 2226.29 m2 g-1 and 1.89 nm, and their N and O contents are 5.08% and 9.4%, respectively, which is beneficial for increasing pseudocapacitance. Furthermore, the doping of the metal Ni increases the graphitization degree of the PICNs and promotes the conversion of pyridine-N to graphitized-N. Therefore, the PICNs possess a high specific capacitance of 407 F g-1 at a current density of 0.5 A g-1, a high capacitance retention of 78.62% even at 20 A g-1, and an outstanding cycling stability (over 93% retention rate after 10,000 charge/discharge cycles). Moreover, an energy density of 36.11 W h kg-1 is achieved at a power density of 517.8 W kg-1 during a two-electrode system test, and a retention rate of 87.5% is obtained after 10,000 cycles. This co-hydrothermal treatment as well as nitrogen-doping approach for preparing porous interconnected carbon from SS not only represents an alternative strategy for carbon-based supercapacitor materials but also provides a new option for the utilization of waste SS.

Adefovir dipivoxil induced hypophosphatemic osteomalacia in chronic hepatitis B: a comparative study of Chinese and foreign case series.

BACKGROUND: Adefovir dipivoxil (ADV)-induced renal tubular dysfunction and hypophosphatemic osteomalacia (HO) have been given great consideration in the past few years. However, no standard guidance is available due to a lack of powerful evidence from appropriate long-term prospective case-control studies and variations in the definition of renal adverse events. The aim of this study is to clarify clinical features of ADV-related HO in Chinese chronic hepatitis B patients with long-term ADV treatment in Chinese and non-Chinese comparative case series. METHODS: Retrieval of case reports was based on Pubmed, CNKI, Wan Fang and VIP databases using the key words adefovir dipivoxil, hypophosphatemia, osteomalacia and Fanconi syndrome. We divided patients into Chinese (C group) and Foreign (F group) groups according to their nationality. Comparisons involving demographics, clinical manifestations, tests, treatment and prognosis were conducted between the two groups. RESULTS: Of the patients screened, 120 Chinese patients were identified in the C group, and 32 non-Chinese patients were identified in the F group. The average age of the C group was younger than that of the F group (51.89 years ±10.96 years versus 56.47 years ±11.36 years, t = - 2.084, P = 0.039). No significant difference was found in gender (male to female, 3.29:1 versus 3:1, χ 2 = 0.039, P = 0.844). Although there was no significant difference in the duration of ADV therapy before ostalgia onset, the C group tended to develop adverse events earlier, by 2-3 years, while the F group developed adverse events at 4-5 years (Z = - 1.517, P = 0.129). Prognosis was good after adjustment of the ADV dose and supplemental administration of phosphate and calcitriol. Time to resolution of tubular dysfunction was commenced at the first month, and Chinese patients were more prone to recover in the first 3 months than non-Chinese patients (91.3% of patients in the C group versus 56.3% in the F group, Z = - 3.013, P = 0.003). CONCLUSIONS: Sufficient attention is required for middle-aged males before and during exposure to long-term ADV therapy, regardless of nationality. The clinical picture, laboratory and radiograph alterations are important clues for those patients and are usually characterized by polyarthralgia, renal tubular dysfunction and mineralization defects. Implementation of an early renal tubular injury index is recommended for patients with higher risk, which would prevent further renal injury.

Effect of spleen tyrosine kinase on nonsmall cell lung cancer.

AIM OF STUDY: To investigate the anti.tumor effect of spleen tyrosine kinase. (Syk) on the human nonsmall cell lung cancer cells in vitro and its mechanism. MATERIALS AND METHODS: In this study, we constructed a eukaryotic expression vector pcDNA3.1D/V5-His-TOPO/Syk and transfected it into human nonsmall cell lung cancer cells A549. Then, we not only analyzed the expression of Syk in transfected cells and its invasion but also the expression of matrix metalloproteinase-9 (MMP-9). RESULTS: The results showed that overexpressed Syk significantly inhibited A549 cell invasive ability by decreasing the expression of MMP-9. CONCLUSION: The overexpressed Syk plays an important role in tumor invasion and metastasis, and a negative role in human nonsmall cell lung cancer cells.

Validation of N-glycan markers that improve the performance of CA19-9 in pancreatic cancer.

Pancreatic cancer (PC) has a high mortality rate because it is usually diagnosed late. Glycosylation of proteins is known to change in tumor cells during the development of PC. The objectives of this study were to identify and validate the diagnostic value of novel biomarkers based on N-glycomic profiling for PC. In total, 217 individuals including subjects with PC, pancreatitis, and healthy controls were divided randomly into a training group (n = 164) and validation groups (n = 53). Serum N-glycomic profiling was analyzed by DSA-FACE. The diagnostic model was constructed based on N-glycan markers with logistic stepwise regression. The diagnostic performance of the model was assessed further in validation cohort. The level of total core fucose residues was increased significantly in PC. Two diagnostic models designated GlycoPCtest and PCmodel (combining GlycoPCtest and CA19-9) were constructed to differentiate PC from normal. The area under the receiver operating characteristic curve (AUC) of PCmodel was higher than that of CA19-9 (0.925 vs. 0.878). The diagnostic models based on N-glycans are new, valuable, noninvasive alternatives for identifying PC. The diagnostic efficacy is improved by combined GlycoPCtest and CA19-9 for the discrimination of patients with PC from healthy controls.

Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone-related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor.

Chondrogenesis and endochondral ossification are precisely controlled by cellular interactions with surrounding matrix proteins and growth factors that mediate cellular signaling pathways. Here, we report that extracellular matrix protein 1 (ECM1) is a previously unrecognized regulator of chondrogenesis. ECM1 is induced in the course of chondrogenesis and its expression in chondrocytes strictly depends on parathyroid hormone-related peptide (PTHrP) signaling pathway. Overexpression of ECM1 suppresses, whereas suppression of ECM1 enhances, chondrocyte differentiation and hypertrophy in vitro and ex vivo In addition, target transgene of ECM1 in chondrocytes or osteoblasts in mice leads to striking defects in cartilage development and endochondral bone formation. Of importance, ECM1 seems to be critical for PTHrP action in chondrogenesis, as blockage of ECM1 nearly abolishes PTHrP regulation of chondrocyte hypertrophy, and overexpression of ECM1 rescues disorganized growth plates of PTHrP-null mice. Furthermore, ECM1 and progranulin chondrogenic growth factor constitute an interaction network and act in concert in the regulation of chondrogenesis.-Kong, L., Zhao, Y.-P., Tian, Q.-Y., Feng, J.-Q., Kobayashi, T., Merregaert, J., Liu, C.-J. Extracellular matrix protein 1, a direct targeting molecule of parathyroid hormone-related peptide, negatively regulates chondrogenesis and endochondral ossification via associating with progranulin growth factor.

IL-17 mediates inflammatory reactions via p38/c-Fos and JNK/c-Jun activation in an AP-1-dependent manner in human nucleus pulposus cells.

BACKGROUND: Low back pain and sciatica caused by intervertebral disc (IVD) disease are associated with inflammatory responses. The cytokine interleukin 17 (IL-17) is elevated in herniated and degenerated IVD tissues and acts as a regulator of disc inflammation. The objective of this study was to investigate the involvement of IL-17A in IVD inflammatory response and to explore the mechanisms underlying this response. METHODS: Cells were isolated from nucleus pulposus (NP) tissues collected from patients undergoing surgeries for IVD degeneration. The concentrations of COX2 and PGE2, as well as of select proteins involved in the mitogen-activated protein kinase (MAPK)/activating protein-1 (AP-1) pathway, were quantified in NP cells after exposure to IL-17 with or without pretreatment with MAPK or AP-1 inhibitors. RESULTS: Our results showed that IL-17A increased COX2 expression and PGE2 production via the activation of MAPKs, including p38 kinase and Jun N-terminal kinase (JNK). Moreover, IL-17A-induced COX2 and PGE2 production was shown to rely on p38/c-Fos and JNK/c-Jun activation in an AP-1-dependent manner. CONCLUSION: In summary, our results indicate that IL-17A enhances COX2 expression and PGE2 production via the p38/c-Fos and JNK/c-Jun signalling pathways in NP cells to mediate IVD inflammation.

Effect of rAAV2-hTGFß1 Gene Transfer on Matrix Synthesis in an In Vivo Rabbit Disk Degeneration Model.

STUDY DESIGN: In vivo gene transfer for disk regeneration. OBJECTIVE: To evaluate the efficiency and effect of human transforming growth factor ß1 (hTGFß1) gene transfer mediated by adeno-associated virus (AAV) in a rabbit disk degeneration model induced by fibronectin fragment (Fn-f). SUMMARY OF BACKGROUND DATA: Gene therapy for disk degeneration has been reported to be effective. Nevertheless, few investigations have targeted the degenerative nucleus pulposus (NP) cells in vivo. Fn-f-induced degeneration has been previously verified to be a useful model for the study of disk degeneration at the molecular level. AAV vector is well suited for gene transfer in the disk for its lower immunogenicity and higher safety. MATERIALS AND METHODS: The early dedifferentiated NP cells were transfected with rAAV2-mediated enhanced green fluorescent protein (EGFP) gene in vitro. Fluorescence expression was observed 48 hours later. The rabbit disk degeneration model was established with a microinjection of Fn-f. Ninety-six degenerative disks of 24 rabbits were injected with rAAV2-hTGFß1 (group A), rAAV2-EGFP (group B), or PBS (group C). Immunohistochemical staining for hTGFß1 and fluorescence observation were performed at the 1- and 12-week time points, respectively. 35S-sulfate incorporation assay and Western blot analysis were used to measure the synthesis of proteoglycan and collagen type II at 4-, 8-, and 12-week time points. RESULTS: The dedifferentiated NP cells exhibited intensive fluorescence expression in vitro, with a transfection rate of 90%. In vivo, disks in group A showed enhanced positive hTGFß1 immunostaining at the 1-week time point. At the 4-, 8-, and 12-week time points, disks in group A exhibited significantly increased proteoglycan and collagen type II synthesis compared with the other 2 groups (P<0.01). Abundant green fluorescence was observed in the disks in group B at the 12-week time point. CONCLUSIONS: Early degenerative NP cells are susceptible to AAV-mediated gene transfer in vitro and in vivo. The rapid and prolonged target protein expressions and increased matrix synthesis indicated that AAV-mediated therapeutic gene transfer can be a promising form of treatment for disk regeneration in vivo.

Progranulin suppresses titanium particle induced inflammatory osteolysis by targeting TNFα signaling.

Aseptic loosening is a major complication of prosthetic joint surgery, characterized by chronic inflammation, pain, and osteolysis surrounding the bone-implant interface. Progranulin (PGRN) is known to have anti-inflammatory action by binding to Tumor Necrosis Factor (TNF) receptors and antagonizing TNFα. Here we report that titanium particles significantly induced PGRN expression in RAW264.7 cells and also in a mouse air-pouch model of inflammation. PGRN-deficiency enhanced, whereas administration of recombinant PGRN effectively inhibited, titanium particle-induced inflammation in an air pouch model. In addition, PGRN also significantly inhibited titanium particle-induced osteoclastogenesis and calvarial osteolysis in vitro, ex vivo and in vivo. Mechanistic studies demonstrated that the inhibition of PGRN on titanium particle induced-inflammation is primarily via neutralizing the titanium particle-activated TNFα/NF-κB signaling pathway and this is evidenced by the suppression of particle-induced IκB phosphorylation, NF-κB p65 nuclear translocation, and activity of the NF-κB-specific reporter gene. Collectively, these findings not only demonstrate that PGRN plays an important role in inhibiting titanium particle-induced inflammation, but also provide a potential therapeutic agent for the prevention of wear debris-induced inflammation and osteolysis.

Reversal of Chemoresistance in Human Lung Cancer Cell Line A549/Taxol by Synthetic Second Mitochondria-Derived Activator of Caspase Peptide.

BACKGROUND: Chemoresistance is a leading cause of treatment failure in advanced lung cancer, including that with the extensively prescribed taxol. Recently, a series of structurally unique second mitochondria-derived activators of caspase (Smac) that act as antagonists of inhibitor of apoptosis proteins (IAPs) have been discovered, exhibiting the ability of inducing enhanced apoptosis of various cancer cell types when combined with chemotherapy. In the present study, we synthesized the second mitochondria-derived activator of caspase peptide (Smac-N7 for short) and explored its capacity in combination with taxol in vitro. METHODS: The sensitivity assay and reversal ability of Smac-N7 were tested by MTT. Flow cytometry was used to analyze apoptosis of cells with Annexin V/PI double staining technique. Cell cloning ability was performed to reflect its biological behavior in each group. RESULTS: Concentrations with inhibitory rates < 10% were selected as the reversal value of Smac-N7 peptide using MTT. The reversal folds were 2.52, 3.26, 3.67, and 5.4 in taxol + Smac-N7 (0.0390625, 0.078125, 0.15625, 0.3125 µg/mL, respectively), and concentrations of Smac-N7 and reversal folds appeared in an obvious positive correlation (r(s) = 1, p = 0.000). Apoptosis analyzed at 48 hours by flow cytometry showed the apoptotic rates in taxol and 0.0390625, 0.078125, 0.15625, and 0.3125 µg/mL Smac-N7 + taxol groups were 15.4 ± 1.09%, 20.8% ± 2.18%, 28.4% ± 4.17%, 37.64% ± 6.41%, and 46.6% ± 7.76%, respectively. Concentrations of Smac-N7 appeared to have negative correlations with PE and SF (r(s) = -1, p < 0.05), which showed that the cells' cloning ability in 0.3125 µg/mL Smac-N7 + taxol group was worse than that of other groups. CONCLUSIONS: When combined with taxol, 0.3125 µg/mL Smac-N7 peptide may significantly increase taxol-induced apoptosis in chemoresistant A549/taxol lung cells at 48 hours, and is potentially useful as a reversal agent in lung cancer therapy.

Surgical Treatment of Cervical Spondylotic Myelopathy Associated Hypertension--A Retrospective Study of 309 Patients.

Hypertension is the most prevalent cardiovascular disease, and various risk factors are known to be involved in it. Cervical spondylotic myelopathy (CSM) is the most common non-traumatic cause of myelopathy, which displays neurological symptoms and may induce systemic symptoms. To date, it is still unknown whether CSM is associated with hypertension, and if so, whether the decompression operations can attenuate CSM associated hypertension. Here, a total of 309 patients with CSM who received anterior or posterior decompression surgery were enrolled as subjects. Blood pressure measurements were performed before and within one week after the surgery. Among the 309 subjects, 144 (46.6%) of them exhibited hypertension before surgery, a significantly higher ratio than that of the whole population. One week after surgery, blood pressure of 106 (73.6%) patients turned back to normal. Blood pressure of another 37(25.7%) patients decreased with different degrees, although still higher than normal. Moreover, it appears that both approaches were effective in improving blood pressure, while the posterior approach was more effective in decreasing systolic blood pressure. We speculate this type of hypertension might result from hyperactivity of sympathetic nervous system as the heart rate of these patients decreased after surgery as well. Collectively, compression of spinal cord in CSM patients might be associated with hypertension, and decompression surgery largely attenuated this type of hypertension. These findings prove CSM to be a potential associated factor of high blood pressure and may shed light on therapies of hypertension in clinics.

Serum N-Glycans: A New Diagnostic Biomarker for Light Chain Multiple Myeloma.

The aim of this study was to evaluate the diagnostic and differential diagnostic power of serum N-glycans for light chain multiple myeloma (LCMM). A total of 167 cases of subjects, including 42 LCMM, 42 IgG myeloma, 41 IgA myeloma, and 42 healthy controls were recruited in this study. DNA sequencer-assisted fluorophore-assisted capillary electrophoresis (DSA-FACE) was applied to determine the quantitive abundance of serum N-glycans. The core fucosylated, bisecting and sialylated modifications were analyzed in serum of LCMM patients (n=20) and healthy controls (n=20) randomly selected from the same cohort by lectin blot. Moreover, serum sialic acid (SA) level was measured by enzymatic method. We found two N-glycan structures (NG1A2F, Peak3; NA2FB, Peak7) showed the optimum diagnostic efficacy with area under the ROC curve (AUC) 0.939 and 0.940 between LCMM and healthy control. The sensitivity and specificity of Peak3 were 88.1% and 92.9%, while Peak7 were 92.9% and 97.6%, respectively. The abundance of Peak3 could differentiate LCMM from IgG myeloma with AUC 0.899, sensitivity 100% and specificity 76.2%, and Peak7 could be used to differentiate LCMM from IgA myeloma with AUC 0.922, sensitivity 92.9% and specificity 82.9%. Serum SA level was significantly higher in patients with LCMM than that in healthy controls. Moreover, the decreased core fucosylation, bisecting and increased sialylation characters of serum glycoproteins were observed in patients with LCMM. We concluded that serum N-glycan could provide a simple, reliable and non-invasive biomarker for LCMM diagnosis and abnormal glycosylation might imply a new potential therapeutic target in LCMM.

ADAMTS-7 exhibits elevated expression in cartilage of osteonecrosis of femoral head and has a positive correlation with TNF- α and NF- κ B P65.

ADAMTS-7 has been reported to exaggerate cartilage degeneration and to be associated with TNF-α and NF-κB signaling pathway. In this study we compared the expression of ADAMTS-7, TNF-α, and Phospho-NF-κB in patients with femoral neck fracture (FNF) and osteonecrosis of femoral head (ONFH) at different stages. We found that expression of ADAMTS-7, TNF-α, and Phospho-NF-κB was significantly upregulated in ONFH patients' articular cartilage and related to the pathogenesis of ONFH. Thus we conclude that ADAMTS-7 level appears to be positively associated with expression of TNF-α and Phospho-NF-κB P65 in cartilage, which may imply its association with cartilage destruction of ONFH.

Progranulin protects against osteoarthritis through interacting with TNF-α and ß-Catenin signalling.

OBJECTIVE: Progranulin (PGRN) was previously isolated as an osteoarthritis (OA)-associated growth factor. Additionally, PGRN was found to play a therapeutic role in inflammatory arthritis mice models through antagonising tumour necrosis factor α (TNF-α). This study was aimed at investigating the role of PGRN in degradation of cartilage and progression of OA. METHODS: Progression of OA was analysed in both spontaneous and surgically induced OA models in wild type and PGRN-deficient mice. Cartilage degradation and OA were evaluated using Safranin O staining, immunohistochemistry and ELISA. Additionally, mRNA expression of degenerative factors and catabolic markers known to be involved in cartilage degeneration in OA were analysed. Furthermore, the anabolic effects and underlying mechanisms of PGRN were investigated by in vitro experiments with primary chondrocytes. RESULTS: Here, we found that deficiency of PGRN led to spontaneous OA-like phenotype in 'aged' mice. Additionally, PGRN-deficient mice exhibited exaggerated breakdown of cartilage structure and OA progression, while local delivery of recombinant PGRN protein attenuated degradation of cartilage matrix and protected against OA development in surgically induced OA models. Furthermore, PGRN activated extracellular signal-regulated kinases (ERK) 1/2 signalling and elevated the levels of anabolic biomarkers in human chondrocyte, and the protective function of PGRN was mediated mainly through TNF receptor 2. Additionally, PGRN suppressed inflammatory action of TNF-α and inhibited the activation of ß-Catenin signalling in cartilage and chondrocytes. CONCLUSIONS: Collectively, this study provides new insight into the pathogenesis of OA, and also presents PGRN as a potential target for the treatment of joint degenerative diseases, including OA.

Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro.

AIM: To evaluate the biochemical features and activities of a glyco-engineered form of the anti-human epidermal growth factor receptor monoclonal antibody (EGFR mAb) cetuximab in vitro. METHODS: The genes encoding the Chinese hamster bisecting glycosylation enzyme (GnTIII) and anti-human EGFR mAb were cloned and coexpressed in CHO DG44 cells. The bisecting-glycosylated recombinant EGFR mAb (bisec-EGFR mAb) produced by these cells was characterized with regard to its glycan profile, antiproliferative activity, Fc receptor binding affinity and cell lysis capability. The content of galactose-α-1,3-galactose (α-Gal) in the bisec-EGFR mAb was measured using HPAEC-PAD. RESULTS: The bisec-EGFR mAb had a higher content of bisecting N-acetylglucosamine residues. Compared to the wild type EGFR mAb, the bisec-EGFR mAb exhibited 3-fold higher cell lysis capability in the antibody-dependent cellular cytotoxicity assay, and 1.36-fold higher antiproliferative activity against the human epidermoid carcinoma line A431. Furthermore, the bisec-EGFR mAb had a higher binding affinity for human FcγRIa and FcγRIIIa-158F than the wild type EGFR mAb. Moreover, α-Gal, which was responsible for cetuximab-induced hypersensitivity reactions, was not detected in the bisec-EGFR mAb. CONCLUSION: The glyco-engineered EGFR mAb with more bisecting modifications and lower α-Gal content than the approved therapeutic antibody Erbitux shows improved functionality in vitro, and requires in vivo validations.

Decreased core-fucosylation contributes to malignancy in gastric cancer.

The object of the study is to identify N-glycan profiling changes associated with gastric cancer and explore the impact of core-fucosylation on biological behaviors of human gastric cancer cells. A total of 244 subjects including gastric cancer, gastric ulcer and healthy control were recruited. N-glycan profiling from serum and total proteins in gastric tissues was analyzed by DNA sequencer-assisted fluorophore-assisted capillary electrophoresis. The abundance of total core-fucosylated residues and the expression of enzymes involved in core-fucosylation were analyzed with lectin blot, quantitative reverse transcription-polymerase chain reaction, western blot, Immunohistochemical staining and lectin-histochemical staining. The recombinant plasmids of GDP-fucose transporter and α-1,6-fucosyltransferase (Fut8) were constructed and transfected into gastric cancer cell lines BGC-823 and SGC-7901. CCK-8 and wound healing assay were used to assess the functional impact of core-fucosylation modulation on cell proliferation and migration. Characteristic serum N-glycan profiles were found in gastric cancer. Compared with the healthy control, a trianntenary structure abundance, peak 9 (NA3Fb), was increased significantly in gastric cancer, while the total abundance of core-fucosylated residues (sumfuc) was decreased. Core-fucosylated structures, peak6(NA2F) and peak7(NA2FB) were deceased in gastric tumor tissues when compared with that in adjacent non-tumor tissues. Consistently, lens culinaris agglutinin (LCA)-binding proteins were decreased significantly in sera of gastric cancer, and protein level of Fut8 was decreased significantly in gastric tumor tissues compared with that in adjacent non-tumor tissues. Upregulation of GDP-Tr and Fut8 could inhibit proliferation, but had no significant influence on migration of BGC-823 and SGC-7901 cells. Core-fucosylation is down regulated in gastric cancer. Upregulation of core-fucosylation could inhibit proliferation of the human gastric cancer cells.

Inhibitory effects of syk transfection on lung cancer cell invasion.

OBJECTIVE: Spleen tyrosine kinase (Syk) is closely related to tumor invasion and metastasis, and has been shown to have potential inhibitory effects in tumors. In this study, we constructed a eukaryotic expression vector for Syk and analyzed its effects on invasive ability of the A549 non-small cell lung cancer cell line in vitro. METHODS: A fragment of Syk was obtained by RT-PCR from human lung cancer cells and cloned into the expression vector pLNCXSyk. After restriction endonuclease digestion, PCR and DNA sequencing confirmation, the recombinant Syk expression plasmid was transfected into A549 human lung cancer cells using lipofectamine protocols. After selection, the cells stably expressed Syk. Detection of Syk expression of the cells by RT-PCR, and invasive ability were examined. RESULTS: The eukaryotic expression plamid pLNCXSyk was constructed and expressed stably in the A549 human lung cancer cells. The RT-PCR results showed that Syk mRNA expression was upregulated significantly (P<0.05). Lower invasion through a basal membrane were apparent after transfection (P<0.05). CONCLUSIONS: A eukaryotic expression plasmid to cause Syk expression in lung cancer cells can obviously inhibit their invasive ability in vitro.

The promotion of bone healing by progranulin, a downstream molecule of BMP-2, through interacting with TNF/TNFR signaling.

Endochondral ossification plays a key role in the bone healing process, which requires normal cartilage callus formation. Progranulin (PGRN) growth factor is known to enhance chondrocyte differentiation and endochondral ossification during development, yet whether PGRN also plays a role in bone regeneration remains unknown. In this study we established surgically-induced bone defect and ectopic bone formation models based on genetically-modified mice. Thereafter, the bone healing process of those mice was analyzed through radiological assays including X-ray and micro CT, and morphological analysis including histology and immunohistochemistry. PGRN deficiency delayed bone healing, while recombinant PGRN enhanced bone regeneration. Moreover, PGRN was required for BMP-2 induction of osteoblastogenesis and ectopic bone formation. Furthermore, the role of PGRN in bone repair was mediated, at least in part, through interacting with TNF-α signaling pathway. PGRN-mediated bone formation depends on TNFR2 but not TNFR1, as PGRN promoted bone regeneration in deficiency of TNFR1 but lost such effect in TNFR2 deficient mice. PGRN blocked TNF-α-induced inflammatory osteoclastogenesis and protected BMP-2-mediated ectopic bone formation in TNF-α transgenic mice. Collectively, PGRN acts as a critical mediator of the bone healing process by constituting an interplay network with BMP-2 and TNF-α signaling, and this represents a potential molecular target for treatment of fractures, especially under inflammatory conditions.