Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

The Rho kinase inhibitor Y-27632 facilitates the differentiation of bone marrow mesenchymal stem cells

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, but technical challenges have limited cell yield and thus the success of these potential treatments. The Rho GTPases and downstream Rho kinases (Rho coiled-coil kinases or ROCKs) are central regulators of cytoskeletal dynamics during the cell cycle and thus help determine the balance between stem cells self-renewal, lineage commitment, and apoptosis. Here, we examined if suppression of ROCK signaling enhances the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) differentiation into neurons and neuroglial cells. BMSCs were cultured in epidermal growth factor (EGF, 10 µg/l) and basic fibroblastic growth factor (bFGF, 10 µg/l) in the presence or absence of the Rho kinase inhibitor Y-27632 (10 µM). The expression levels of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence and Western blotting. The average number of NSE-positive cells increased from 83.20 ± 8.677 (positive ratio 0.2140 ± 0.0119) to 109.20 ± 8.430 (positive ratio 0.3193 ± 0.0161) per visual field in the presence of Y-27632, while GFAP-positive cell number increased from 96.30 ± 8.486 (positive ratio 0.18 ± 0.0152) to 107.50 ± 8.683 (positive ratio 0.27 ± 0.0115) (P < 0.05 for both). Both NSE and GFAP protein expression levels were enhanced significantly by Y-27632 treatment (NSE: 0.74 ± 0.05 vs. 1.03 ± 0.06; GFAP: 0.64 ± 0.08 vs. 0.97 ± 0.05, both P < 0.01) as indicated by Western blots. The Rho kinase inhibitor Y-27632 concomitant with EGF and bFGF stimulation promotes BMSC differentiation into neural cells. Control of Rho kinase activity may enhance the efficiency of stem cell-based treatments for neurodegenerative diseases.     

Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.     

Transarterial chemoembolization combined with sorafenib for unresectable hepatocellular carcinoma: a systematic review and meta-analysis

Sorafenib in combination with Transarterial chemoembolization (TACE) is increasingly used in patients with unresectable hepatocellular carcinoma (HCC), but the current evidence is still controversial. The aim of this systematic review was to evaluate the effectiveness and safety of TACE plus sorafenib versus TACE alone for unresectable HCC. We searched PubMed, EMBASE and the Cochrane Library for clinical trials comparing TACE plus sorafenib with TACE alone for unresectable HCC. The study outcomes included overall survival (OS), time to progression (TTP), objective response and adverse events (AEs). Six studies including 1,181 patients were included. Meta-analysis of all studies suggested that the combination therapy group had significant longer OS than TACE group [hazard ratio (HR) = 0.64, 95 % confidence interval (CI) = 0.43–0.97], but the pooled HR of randomized controlled trials (RCTs) failed to achieve statistical significance. For TTP, meta-analysis in both RCTs subgroup and retrospective studies subgroup suggested that combination therapy was superior to TACE group. The combination therapy was also associated with better response to treatment (risk ratio = 1.45, 95 % CI = 1.04–2.02) when both RCTs and retrospective studies were pooled. However, the sorafenib associated AEs were more frequent in the combination therapy group. In conclusion, the combination of TACE and sorafenib is likely to improve OS, TTP and response to treatment when compared with TACE monotherapy. The combination group is also associated with more sorafenib-related AEs.     

Preparation of cinnamoyl-CoA and analysis of the enzyme activity of recombinant CCR protein from Populus tomentosa

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), which catalyzes the reduction of cinnamoyl-CoA esters to their respective cinnamaldehydes, is considered as a key enzyme in lignin formation. The substrates of CCR, cinnamoyl-CoA esters, are products of 4-Coumarate-CoA ligase (4CL, EC 6.2.1.12), which is an enzyme upstream of CCR. The PtCCR and Pt4CL were isolated from Populus tomentosa and expressed in E. coli. Results showed that 4CL can catalyze the conversion of hydroxycinnamic acids to cinnamoyl-CoA esters, with high efficiency. The purification of esters using SPE cartridges suggested that 40 % methanol with 0.1 M of acetic acid was the optimal elution buffer for cinnamoyl-CoA esters. The optimization of prokaryotic expression demonstrated that the best expression conditions for recombinant PtCCR was 6 h of 0.4 mM IPTG induction at 37 °C. PtCCR enzyme assay illustrated that the recombinant protein can catalyze the reduction of cinnamoyl-CoA esters. Kinetics analysis showed that feruloyl-CoA has higher affinity to PtCCR with faster reaction speed (Vmax), indicating that feruloyl-CoA was the most favorable substrate for PtCCR catalysis. The recombinant protein was expressed in E. coli, purified through affinity column chromatography, and characterized by SDS-PAGE. SPE cartridges were used to purify the ester products of the Pt4CL reaction. HPLC-MS was used to analyze the structure of esters and evaluate their purity or quantity. Furthermore, the enzyme activity of recombinant CCR to feruloyl-CoA at different pHs indicated that compartmentalization may be an important factor in lignin monomer formation.     

Expression of GroES TB antigen in tobacco and potato

As the world races towards a plant-based bioeconomy, plants known to be ideal and economical bioreactors are being harnessed for the production of recombinant proteins. The major immunodominant 10 kDa GroES TB antigen (Chaperonin 10) gene from Mycobacterium tuberculosis was selected for expression in plants as a putative tuberculosis (TB) subunit vaccine candidate. Two crops, tobacco and potato, were engineered by stable plant transformation for expression of the 10 kDa GroES TB antigen using non-viral binary vectors. The integration of the GroES TB gene into the genomes of tobacco and potato was confirmed by PCR and Southern blotting. The expression of the GroES TB antigen in tobacco was 0.04–1.2 % of the total soluble protein (TSP). However, the expression of the same TB antigen in the Indian potato cv. Kufri bahar was comparatively low (0.033 % of TSP). The recombinant GroES plant derived protein was characterised and confirmed by MALDI-TOF–TOF and ELISA. This is the first report of the expression of the 10 kDa chaperonin in tobacco and potato.     

Apparent Reduction of ADAM10 in Scrapie-Infected Cultured Cells and in the Brains of Scrapie-Infected Rodents

It has been described that A disintegrin and metalloproteinase (ADAM10) may involve in the physiopathology of prion diseases, but the direct molecular basis still remains unsolved. In this study, we confirmed that ADAM10 was able to cleave recombinant human prion protein in vitro. Using immunoprecipitation tests (IP) and immunofluorescent assays (IFA), reliable molecular interaction between the native cellular form of PrP (PrP^C) and ADAM10 was observed not only in various cultured neuronal cell lines but also in brain homogenates of healthy hamsters and mice. Only mature ADAM10 (after removal of its prodomain) molecules showed the binding activity with the native PrP^C. Remarkably more prion protein (PrP)-ADAM10 complexes were detected in the membrane fraction of cultured cells. In the scrapie-infected SMB cell model, the endogenous ADAM10 levels, especially the mature ADAM10, were significantly decreased in the fraction of cell membrane. IP and IFA tests of prion-infected SMB-S15 cells confirmed no detectable PrP-ADAM10 complex in the cellular lysates and PrP-ADAM10 co-localization on the cell surface. Furthermore, we demonstrated that the levels of ADAM10 in the brain homogenates of scrapie agent 263K-infected hamsters and agent ME7-infected mice were also almost diminished at the terminal stage, showing time-dependent decreases during the incubation period. Our data here provide the solid molecular basis for the endoproteolysis of ADAM10 on PrP molecules and interaction between ADAM10 and PrP^C. Obvious loss of ADAM10 during prion infection in vitro and in vivo highlights that ADAM10 may play essential pathophysiological roles in prion replication and accumulation.     

Expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the Spinal Tuberculous Focus and Its Impact on the Disease

To investigate the expression of TNF-α, IFN-γ, TGF-β, and IL-4 in the spinal tuberculous focus and its relationship with the lesions type, severity, and bone destruction. The pathological samples of patients with spinal tuberculosis (TB) were divided into hyperplasia group and necrosis group according to their intra-operative and post-operative pathological findings. Normal bone tissues were taken as the control group. Pathology and expression of TNF-α, IFN-γ, TGF-β, and IL-4 in different tissues were compared among these three groups using immunohistochemical staining, quantitative image analysis, and measurement of bone tissue. 286 granulomas observed in the 14 samples in the hyperplasia group, which included 84 necrotizing and 202 non-necrotizing granulomas. As for the 20 samples in the necrosis group, there were 356 necrotizing and 186 non-necrotizing granulomas among all the 542 granulomas. The proportion of necrotizing granulomas in the necrosis group was significantly higher than that of the hyperplasia group. By inter-group comparison, expression of TNF-α, IFN-γ of granulomas in the hyperplasia group was significantly higher than that of the necrosis group, while the expression of TGF-β, IL-4 of granulomas in the necrosis group was significantly higher than that of the hyperplasia group. Also, expression of IFN-γ of non-necrotizing granulomas was significantly higher than that of necrotizing granulomas in the hyperplasia group, and expression of TGF-β in necrotizing granulomas was significantly higher than that of non-necrotizing granulomas in the necrosis group. The lesions were mainly bone resorption in the hyperplasia group, whereas mostly necrotic bones accompanied by local fibrosis in the necrosis group. Expression levels of TNF-α, IFN-γ in the hyperplasia group have a positive correlation to bone loss, whereas expression levels of TGF-β, IL-4 in the necrosis group have a positive correlation to the bone formation. The high expressions of TNF-α, IFN-γ in the spinal tuberculous focus were associated with protective immune cells. TGF-β and IL-4 were related to allergic lesions, fibrosis and osteogenesis. Expression imbalance of TNF-α, IFN-γ, TGF-β, and IL-4 might aggravate the allergy of TB.     

The Significance and Mechanism of Propofol on Treatment of Ischemia Reperfusion Induced Lung Injury in Rats

This study is aimed to investigate the efficacy and underlying the mechanism of propofol in treatment of ischemia reperfusion (IR)-induced lung injury in rats, providing a novel insight of therapeutic strategy for IR-induced lung injury. 120 healthy SD rats were selected and randomly divided into sham operation group, IR group, and propofol group (40 rats per group). Bronchoalveolar lavage fluid (BALF) protein content, serum protein content, lung permeability index, lung water content rate, methane dicarboxylic aldehyde (MDA) in lung tissue, superoxide dismutase (SOD), nitric oxide (NO), endothelin (ET-1), toll-like receptor 4 (TLR4), nuclear factor (NF-κB), and tumor necrosis factor-α (TNF-α) were examined and compared among different groups to evaluate the therapeutical effects of propofol on IR-induced lung injury and analyze the mechanism. In sham operation group, neither change in lung tissue nor pulmonary interstitial edema or alveolar wall damage was found under microscope; in IR group, marked pulmonary interstitial edema and alveolar wall damage complicated with inflammatory cell infiltration and hemorrhage were found; in propofol group, alveolar wall widening was observed, however, hemorrhage in alveolar cavity, inflammatory infiltration and tissue damage were less significant than in IR group. At 3 h after reperfusion, BALF protein content, lung permeability index, and lung water content rate were all significantly increased in IR group and propofol group, while the serum protein content was significantly lower than sham operation group (p < 0.05). Moreover, we found that the change of above parameters in propofol group was less significant than in IR group (p < 0.05). No statistically significant difference was found in ET-1 levels in different groups (p > 0.05). In contrast, MDA and NO in IR group and propofol group were significantly increased, while SOD activity was significantly decreased (p < 0.05). Furthermore, the change of above parameters in propofol group was less significant than in IR group (p < 0.05). In addition, mRNAs of TLR4, NF-κB, and TNF-α were significantly increased in IR group and propofol group (p < 0.05) with more significant change in IR group compared with propofol group (p < 0.05). Propofol has protective effects against IR-induced lung injury by improving activity of oxygen radical and restoring NO/ET-1 dynamic balance. Besides, regulation of TLR4, NF-κB, and TNF-α by propofol also play important role in alleviating IR-induced lung injury.     

Modulation of inducible nitric oxide synthase (iNOS) expression and cardiovascular responses during static exercise following iNOS antagonism within the ventrolateral medulla

Previous reports indicate that inducible nitric oxide synthase (iNOS) blockade within the rostral ventrolateral medulla (RVLM) and caudal ventrolateral medulla (CVLM) differentially modulated cardiovascular responses, medullary glutamate, and GABA concentrations during static skeletal muscle contraction. In the current study, we determined the role of iNOS antagonism within the RVLM and CVLM on cardiovascular responses and iNOS protein expression during the exercise pressor reflex in anesthetized rats. Following 120 min of bilateral microdialysis of a selective iNOS antagonist, aminoguanidine (AGN; 10 µM), into the RVLM, the pressor responses were attenuated by 72 % and changes in heart rate were reduced by 38 % during a static muscle contraction. Furthermore, western blot analysis of iNOS protein abundance within the RVLM revealed a significant attenuation when compared to control animals. In contrast, bilateral administration of AGN (10 µM) into the CVLM augmented the increases in mean arterial pressure by 60 % and potentiated changes in heart rate by 61 % during muscle contractions, but did not alter expression of the iNOS protein within the CVLM. These results demonstrate that iNOS protein expression within the ventrolateral medulla is differentially regulated by iNOS blockade that may, in part, contribute to the modulation of cardiovascular responses during static exercise.     

Quercetin Potentiates the Antitumor Activity of Rituximab in Diffuse Large B-Cell Lymphoma by Inhibiting STAT3 Pathway

STAT3 pathway plays an important role in the growth of diffuse large B-cell lymphoma (DLBCL) cells. Here we investigated the antitumor activity of Quercetin, a flavonoid compound, in combination with rituximab in DLBCL cell lines in vitro. We found that Quercetin synergistically enhanced rituximab-induced growth inhibition and apoptosis in DLBCL cell lines. Moreover, we found Quercetin exerted inhibitory activity against STAT3 pathway and downregulated the expression of survival genes. These results suggest that combining the Quercetin with rituximab may present an attractive and potentially effective way for the treatment of DLBCL.     

Biological characteristics of rat dorsal root ganglion cell and human vascular endothelial cell in mono- and co-culture

This study aimed to evaluate the biological activity of rat dorsal root ganglion cell (DRGC) and human vascular endothelial cell (HMVEC) in mono- and co-culture. Expression levels of vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) mRNA were measured by quantitative real-time RT-PCR (qRT-PCR). Western blot analysis was used to identify VEGF and NGF protein expressions. Cell injury was assessed by measuring cell viability with methylthiazol tetrazolium (MTT) assay. The results showed that VEGF and NGF mRNA levels in the HMVEC+DRGC group were significantly higher than those in the DRGC and HMVEC groups (all p < 0.05). There were also greater increases in both VEGF and NGF protein expressions in the HMVEC+DRGC group than those in the DRGC and HMVEC groups (all p < 0.05). The results of MTT analysis revealed significant differences in cell viability among the HMVEC+DRGC group and the DRGC and HMVEC groups (all p < 0.05). In summary, our findings provide evidence that DRGC and HMVEC in co-culture may exhibit greater biological activity than DRGC in mono-culture.     

Immune Response of the VEGF/bFGF Complex Peptide Vaccine and Function of Immune Antibodies in Inhibiting Migration of HUVEC Cells and Proliferation of Cancer Cells

Overexpression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis and metastasis in tumors. VEGF/bFGF complex peptide (VBP3) was designed to elicit the body to produce both high titer anti-VEGF and anti-bFGF antibodies to inhibit tumor angiogenesis and tumor growth. BALB/c mice were immunized with the VEGF/bFGF complex peptide, and the immune responses were assayed. Splenocytes were separated from the immunized mice and the CD4, CD8 T cells and IFN-γ were assayed by Flow cytometry. The results showed that the VBP3 could effectively stimulate immune response in mice and resulted in the increase of CD4 and CD8 T cells. CD4+ T cells and CD8+ T cells were increased from 10.78 to 15.13 and 6.82 to 11.58 % respectively. Polyclonal antibodies purified from the VBP3 immunized mice showed good anti-proliferation function to lung cancer cells, and resulted in the decrease of phosphroylation level of Akt and Erk assayed by the Western-blot. Transwell assays showed that the migration of HUVEC cells was inhibited by the antibodies. The results revealed that the VBP3 have good immunogenicity and may be used as a vaccine for tumor therapy.     

The amino-terminal sequence ofStreptomyces griseus carboxypeptidase

The amino-terminal sequence of carboxypeptidase fromStreptomyces griseus was determined using a new protocol for automatic Edman degradation that reduced background noise. The sequence of the first 48 residues is: Asp-Phe-Pro-Pro-Ala-Asp-Ser-Arg-Tyr-His-Asn-Tyr-Ala-Glu-Met-Asn-Ala-Ala-Ile-Asp-Ala-Arg-Ile-Ala-Ala-Asn-Pro-Ser-Ile-Met-Ser-Lys-Arg-Val-Ile-Gly-Lys-Thr-Tyr-Gln-Gly-(Arg)-Asp-Val-Ile-Ala-Val-Lys, which is homologous to that of other zinc-containing carboxypeptidase from vertebrate and invertebrate sources.     

A prepared anti-MSTN polyclonal antibody reverses insulin resistance of diet-induced obese rats via regulation of PI3K/Akt/mTOR&FoxO1 signal pathways

Suppression of myostatin (MSTN) is associated with skeletal muscle atrophy and insulin resistance. However, the mechanisms by which MSTN regulates insulin resistance are not well known. We have explored the signaling pathways through which MSTN regulates insulin resistance in diet-induced obese rats using a polyclonal antibody for MSTN. The anti-MSTN polyclonal antibody significantly improved insulin resistance and whole-body insulin sensitivity, decreased MSTN protein expression in muscle samples by 39 % in diet-induced obese rats. Furthermore, the anti-MSTN polyclonal antibody significantly enhanced PI3K activity (140 %), Akt phosphorylation (86 %), GLUT4 protein expression (23 %), the phosphorylation of mTOR (21 %), and inhibited the phosphorylation of FoxO1 (57 %), but did not affect the phosphorylation of GSK-3β. Thus, suppression of MSTN by the anti-MSTN polyclonal antibody reverses insulin resistance of diet-induced obesity via MSTN/PI3K/Akt/mTOR and MSTN/PI3K/Akt/FoxO1 signaling pathways.     

Modulation,Bioinformatic Screening,and Assessment of Small Molecular Peptides Targeting the Vascular Endothelial Growth Factor Receptor

Vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) are important factors in tumor growth and metastasis. Molecular probes or drugs designed to target VEGF/VEGFR interactions are crucial in tumor molecular imaging and targeted therapy. Bioinformatic methods enable molecular design based on the structure of bio-macromolecules and their interactions. This study was aimed to identify tumor-targeting small-molecule peptides with high affinity for VEGFR using bioinformatics screening. The VEGFR extracellular immunoglobulin-like modules Ig1–Ig3 were used as the target to systematically alter the primary peptide sequence of VEGF_125–136. Molecular docking and surface functional group interaction methods were combined in an in silico screen for polypeptides, which in theory, would have higher affinities for VEGFR. In vitro receptor competition binding assays were used to assess the affinity of the putative VEGFR-binding polypeptides. Rhodamine-conjugated peptides were used to label and visualize peptide-binding sites on A549 cells. Using bioinformatic screening, we identified 20 polypeptides with potentially higher affinity for VEGFR. The polypeptides were capable of inhibiting the binding of ¹²⁵I-VEGF to VEGFR in a dose-dependent manner. The IC₅₀ values of QKRKRKKSRKKH and RKRKRKKSRYIVLS (80 and 185 nmol/L, respectively) were significantly lower than that of VEGF_125–136 (464 nmol/L); thus, the affinity of these peptides for VEGFR was 6- and 2.5-fold higher, respectively, than that of VEGF_125–136. Rhodamine labeling of A549 cells revealed peptide binding mainly on the plasma membrane and in the cytoplasm. Bioinformatic approaches hold promise for the development of molecular imaging probes. Using this approach, we designed two peptides that showed higher affinity toward VEGFR. These polypeptides may be used as molecular probes or drugs targeting VEGFR, which can be utilized in molecular imaging and targeted therapy of certain tumors.