Complement Receptor 3 Pathway and NMDA Receptor 2B Subunit Involve Neuropathic Pain Associated with Spinal Cord Injury.

Background: Neuropathic pain (NP) after spinal cord injury (SCI-evoked NP) is clinically challenging; the underlying mechanisms are not fully understood, leading to a lack of promising treatment options. NP occurs in only a subset of patients with SCI. The injured spinal cord exhibits a series of histopathological changes, and the complement system has been shown to play an important role in these processes. In addition, NMDA receptor subunit 2B (NR2B) is involved in the development and maintenance of NP. This preliminary study was performed to investigate the correlations of the complement receptor 3/complement component 3 (CR3/C3) pathway and NR2B with SCI-evoked NP. Methods: A trauma-induced SCI animal model was established and SCI-evoked NP was evaluated by behavioural analysis. Transcriptome analysis was performed to identify genes in the CR3/C3 pathway related to synaptic modification, while the expression and distribution of NR2B in the injured spinal cord, and the relation to NP, were examined by immunohistochemical analysis. Results: Nine of seventeen SCI rats (52.9%) developed NP. C3 mRNA expression was significantly decreased in SCI-evoked NP rats and significantly increased in the non-NP SCI rats. C1q mRNA and CR3 mRNA expression were significantly increased in all SCI rats, but higher levels of expression were observed in the non-NP SCI rats. NR2B mRNA expression was significantly increased in the SCI-evoked NP rats and significantly decreased in the non-NP SCI rats. In addition, significantly elevated expression of NR2B-positive cells was seen in lamina II of the superficial dorsal horn in SCI-evoked NP rats in comparison with non-NP SCI rats. Conclusion: NP occurred in only a subset of SCI rats, and the CR3/C3 pathway and NR2B were involved in SCI-evoked NP. Further studies are required to determine the mechanisms underlying the SCI-evoked NP associated with the CR3/C3 pathway and NR2B.

Inhibiting the Activity of ABCG2 by KU55933 in Colorectal Cancer.

BACKGROUND: Therapeutic resistance is a frequent problem of cancer treatment and a leading cause of mortality in patients with metastatic colorectal cancer (CRC). Recent insight into the mechanisms that confer multidrug resistance has elucidated that the ATP-binding cassette (ABC) superfamily G member 2 (ABCG2) assists cancer cells in escaping therapeutic stress caused by toxic chemotherapy. Therefore, it is necessary to develop ABCG2 inhibitors. OBJECTIVES: In the present study, we investigated the inhibitory effect of KU55933 on ABCG2 in CRC. METHODS: The cytotoxicity assay and drug accumulation assay were used to examine the inhibitory effect of KU55933 on ABCG2. The protein expressions were detected by Western blot assay. The docking assay was performed to predict the binding site and intermolecular interactions between KU55933 and ABCG2. RESULTS: KU55933 was more potent than the known ABCG2 inhibitor fumitremorgin C to enhance the sensitivity of mitoxantrone and doxorubicin and the intracellular accumulation of mitoxantrone, doxorubicin and rhodamine 123 inside CRC cells with ABCG2 overexpression. Moreover, KU55933 did not affect the protein level of ABCG2. Furthermore, the docking data showed that KU55933 was tightly located in the drug-binding pocket of ABCG2. CONCLUSION: In summary, our data presented that KU55933 could effectively inhibit the drug pump activity of ABCG2 in colorectal cancer, which is further supported by the predicted model that showed the hydrophobic interactions of KU55933 within the drug-binding pocket of ABCG2. KU55933 can potently inhibit the activity of ABCG2 in CRC.

AZ32 Reverses ABCG2-Mediated Multidrug Resistance in Colorectal Cancer.

Colorectal cancer is a common malignancy with the third highest incidence and second highest mortality rate among all cancers in the world. Chemotherapy resistance in colorectal cancer is an essential factor leading to the high mortality rate. The ATP-binding cassette (ABC) superfamily G member 2 (ABCG2) confers multidrug resistance (MDR) to a range of chemotherapeutic agents by decreasing their intracellular content. The development of novel ABCG2 inhibitors has emerged as a tractable strategy to circumvent drug resistance. In this study, an ABCG2-knockout colorectal cancer cell line was established to assist inhibitor screening. Additionally, we found that ataxia-telangiectasia mutated (ATM) kinase inhibitor AZ32 could sensitize ABCG2-overexpressing colorectal cancer cells to ABCG2 substrate chemotherapeutic drugs mitoxantrone and doxorubicin by retaining them inside cells. Western blot assay showed that AZ32 did not alter the expression of ABCG2. Moreover, molecule docking analysis predicted that AZ32 stably located in the transmembrane domain of ABCG2. In conclusion, our result demonstrated that AZ32 could potently reverse ABCG2-mediated MDR in colorectal cancer.

Skipping of exon 10 in Axl pre-mRNA regulated by PTBP1 mediates invasion and metastasis process of liver cancer cells.

The Axl gene is known to encode for a receptor tyrosine kinase involved in the metastasis process of cancer. In this study, we investigated the underlying molecular mechanism of Axl alternative splicing. Methods: The expression levels of PTBP1 in hepatocellular carcinoma (HCC) tissues were obtained from TCGA samples and cell lines. The effect of Axl-L, Axl-S, and PTBP1 on cell growth, migration, invasion tumor formation, and metastasis of liver cancer cells were measured by cell proliferation, wound-healing, invasion, xenograft tumor formation, and metastasis. Interaction between PTBP1 and Axl was explored using cross-link immunoprecipitation, RNA pull-down assays and RNA immunoprecipitation assays. Results: Knockdown of the PTBP1 and exon 10 skipping isoform of Axl (Axl-S), led to impaired invasion and metastasis in hepatoma cells. Immunoprecipitation results indicated that Axl-S protein binds more robustly with Gas6 ligand than Axl-L (exon 10 including) and is more capable of promoting phosphorylation of ERK and AKT proteins. Furthermore, cross-link immunoprecipitation and RNA-pulldown assays revealed that PTBP1 binds to the polypyrimidine sequence(TCCTCTCTGTCCTTTCTTC) on Axl-Intron 9. MS2-GFP-IP experiments demonstrated that PTBP1 competes with U2AF2 for binding to the aforementioned polypyrimidine sequence, thereby inhibiting alternative splicing and ultimately promoting Axl-S production. Conclusion: Our results highlight the biological significance of Axl-S and PTBP1 in tumor metastasis, and show that PTBP1 affects the invasion and metastasis of hepatoma cells by modulating the alternative splicing of Axl exon 10.

Therapeutic effects of the rhSOD2-Hirudin fusion protein on bleomycin-induced pulmonary fibrosis in mice.

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and high mortality, posing a major threat to human health. Increased levels of inflammatory cytokines, reactive oxygen species and coagulation cascade have been extensively reported in IPF. We previously fused Hirudin and human manganese superoxide dismutase (hSOD2) to generate a dual-feature fusion protein, denoted as rhSOD2-Hirudin fusion protein. In this study, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and Hydroxyproline (HYP) assays were used to investigate the effects of rhSOD2-Hirudin protein on thrombin-induced fibroblast proliferation and collagen accumulation in vitro. Subsequently, the mice model of pulmonary fibrosis induced by bleomycin was used for evaluating the anti-inflammatory and anti-fibrotic effects of rhSOD2-Hirudin protein in vivo. Results showed that rhSOD2-Hirudin protein could inhibit the proliferation of fibroblasts and reduce the HYP production in vitro by inhibiting the activity of thrombin. In vivo experiments showed that lung inflammation and fibrosis were significantly decreased in rhSOD2-Hirudin protein-treated mice. Furthermore, rhSOD2-Hirudin protein treatment reduced profibrotic protein and gene expression while reducing the number of inflammatory cells in the lung. In conclusion, rhSOD2-Hirudin protein can effectively attenuate pulmonary fibrosis in vitro and in vivo, mainly by inhibiting the activity of thrombin meanwhile increasing SOD2 levels prevent cells from being damaged by reactive oxygen species, thereby mitigating IPF progression. This study provided important information on the feasibility and efficacy of rhSOD2-Hirudin protein as a novel therapeutic agent for IPF.

An HTML5-Based Pure Website Solution for Rapidly Viewing and Processing Large-Scale 3D Medical Volume Reconstruction on Mobile Internet.

This study aimed to propose a pure web-based solution to serve users to access large-scale 3D medical volume anywhere with good user experience and complete details. A novel solution of the Master-Slave interaction mode was proposed, which absorbed advantages of remote volume rendering and surface rendering. On server side, we designed a message-responding mechanism to listen to interactive requests from clients (Slave model) and to guide Master volume rendering. On client side, we used HTML5 to normalize user-interactive behaviors on Slave model and enhance the accuracy of behavior request and user-friendly experience. The results showed that more than four independent tasks (each with a data size of 249.4 MB) could be simultaneously carried out with a 100-KBps client bandwidth (extreme test); the first loading time was <12 s, and the response time of each behavior request for final high quality image remained at approximately 1 s, while the peak value of bandwidth was <50-KBps. Meanwhile, the FPS value for each client was ≥40. This solution could serve the users by rapidly accessing the application via one URL hyperlink without special software and hardware requirement in a diversified network environment and could be easily integrated into other telemedical systems seamlessly.

The Anti-inflammatory Effect of the CXCR4 Antagonist-N15P Peptide and Its Modulation on Inflammation-Associated Mediators in LPS-Induced PBMC.

Inflammation was the important pathological process of many disease developments, but current therapeutic means for inflammatory diseases are not satisfactory. Chemokines and their receptors represent valuable targets for anti-inflammatory drug discovery. The N15P polypeptide (sequence: LGASWHRPDKCCLGY) is independently developed by our research group, it is a new CXCR4 antagonist drug derived from viral macrophage inflammatory protein-II (vMIP-II). This study aims to clarify the anti-inflammatory potency of N15P polypeptide on the lipopolysaccharide (LPS)-induced inflammation in vitro. In this study, we evaluated the anti-inflammatory effects of N15P polypeptide by the LPS-induced peripheral blood mononuclear cell (PBMC) model and measured the level of inflammatory factors (tumor necrosis factor alpha (TNF-α), IL-6, IL-8, nuclear factor kappaB (NF-κB), cyclooxygenase-2 (COX-2), Toll-like receptor 4 (TLR4), MyD88, phosphoinositide 3-kinase (PI3K), and Akt). The messenger RNA (mRNA) expressions of inflammatory factors were analyzed by real-time PCR (RT-PCR) microarray analysis, and the production of inflammatory factors was measured further by enzyme-linked immunosorbent assay (ELISA) and Western blot. The results showed that the expression of inflammatory factors (TNF-α, IL-6, IL-8, NF-κB, COX-2, TLR4, MyD88, PI3K, and Akt) was downregulated by N15P peptide, suggesting that N15P peptide has a strong inhibitory effect on the inflammatory responses induced by LPS.

A biologically active vMIP-II-IgG3-TfN fusion protein, secreted from methylotrophic yeast Pichia pastoris.

The viral macrophage inflammatory protein II (vMIP-II) which showed a broad-spectrum interaction with both CC and CXC chemokine receptors including CCR5 and CXCR4, two principal coreceptors for the cell entry of human immunodeficiency virus. To explore the feasibility of using TfN as a carrier moiety for delivery of therapeutic proteins, a genetically engineered vMIP-II-IgG3-TfN fusion gene was loaded into the yeast expression vector pPICZα. The linearized recombinant plasmid pPICZα-vMIP-II-IgG3-TfN was transformed into X33 competent cells. The recombinant protein was expressed in methylotrophic yeast Pichia pastoris and was confirmed to have expected molecular mass of 48 kDa by SDS-PAGE. Using methods combining ammonium sulfate precipitation, dialysis, ultrafiltration and affinity chromatography, the vMIP-II-IgG3-TfN fusion protein was successfully purified from the supernatant of the broth. Western-blotting analysis showed that 6× His antibody recognized the purified vMIP-II-IgG3-TfN. CD spectrum revealed a positive peak at 196.5 nm and a negative peak at 209 nm. MALDI-TOF MS analysis showed that the purified vMIP-II-IgG3-TfN was an intact and homogeneous protein. The pepsin digestibility assay showed that the vMIP-II-IgG3-TfN fusion protein could be digested into small fragments by pepsin after 2 min treatment. The vMIP-II-IgG3-TfN fusion protein was found to be stable in human plasma for up to 48 h. Furthermore, in vitro bioactivity assay indicated that the vMIP-II-IgG3-TfN fusion protein can block the chemotaxis of U937 cells induced by SDF1α. In total, this study illustrates the development of an active vMIP-II-IgG3-TfN fusion protein expressed in P. pastoris.

Overexpression of malic enzyme (ME) of Mucor circinelloides improved lipid accumulation in engineered Rhodotorula glutinis.

The oleaginous yeast Rhodotorula glutinis has been known to be a potential feedstock for lipid production. In the present study, we investigated the enhancement of expression of malic enzyme (ME; NADP(+) dependent; EC 1.1.1.40) from Mucor circinelloides as a strategy to improve lipid content inside the yeast cells. The 26S rDNA and 5.8S rDNA gene fragments isolated from Rhodotorula glutinis were used for homologous integration of ME gene into R. glutinis chromosome under the control of the constitutively highly expressed gene phosphoglycerate kinase 1 to achieve stable expression. We demonstrated that by increasing the expression of the foreign ME gene in R. glutinis, we successfully improved the lipid content by more than twofold. At the end of lipid accumulation phrase (96 h) in the transformants, activity of ME was increased by twofold and lipid content of the yeast cells was increased from 18.74 % of the biomass to 39.35 %. Simultaneously, there were no significant differences in fatty acid profiles between the wild-type strain and the recombinant strain. Over 94 % of total fatty acids were C16:0, C18:0, C16:1, C18:1, and C18:2. Our results indicated that heterologous expression of NADP(+)-dependent ME involved in fatty acid biosynthesis indeed increased the lipid accumulation in the oleaginous yeast R. glutinis.

Prediction of novel genes associated with negative regulators of toll-like receptors-induced inflammation based on endotoxin tolerance.

Prior exposure of innate immune cells to lipopolysaccharide (LPS) has caused them to be refractory to further endotoxin stimulation, also termed endotoxin tolerance (ET). Bacterial LPS signals through Toll-like receptor (TLR) 4, which was thought to enable the innate immune system to deal with invasive pathogens and to restrain systemic inflammation efficiently. We established a robust model of ET and determined the level of TNF-α and IL-6 in cultured human monocytes. Then, microarray assay was applied to assess gene expression in this model. The results showed that 356 non-tolerizable genes were differentially expressed at a high level in tolerant monocytes. The genes selected were classified into several categories based on gene ontology (GO) and KEGG pathway database. And then literature annotations, protein-protein interaction (PPI) network, and functional consistency were applied to analyze the non-tolerizable genes. Finally, the microarray results were verified by quantitative real-time PCR of seven representative genes, including the two candidate genes, Spry2 and Smurf2, which were supposed to play a critical role in TLRs-induced inflammation based on literature retrieval. Our results would provide useful information for further analysis of regulating TLRs-induced inflammation, and would facilitate the study of associated mechanisms.

Anti-HCMV and KSHV effect of a trapping ligand antagonist for herpesvirus-encoded GPCR.

We have found and synthesized a trapping ligand peptide H22-LP (the conservative sequence is NAHCALL) from a random phage library according to the broad-spectrum trapping receptor H22, which derived from the residue 14-35 near the N-terminal region of receptor US28 on HCMV. In this study, we will evaluate its potential as an efficient antagonist of US28 and the anti-virus activity, acting as a broad spectrum chemokine receptors antagonist. Stable expression of US28 and ORF74 in NIH/3T3 cells were successfully constructed in vitro. Flow cytomety was used to determine the concentration of Ca2+ induced by H22-LP, and the binding of H22-LP and US28 was confirmed by enzyme-linked immunosorbent assay (ELISA). Antivirus activity of H22-LP on HCMV and KSHV was evaluated by anti-virus experiments. Our data suggest that H22-LP is an effectual antagonist of receptor US28 of HCMV and ORF74 of KSHV in the transfection assay, and it has potential to inhibit infection of HCMV and KSHV. These results provide support for the development of anti-virus strategies based on targeted inhibiting the infection of herpesvirus.

[The viral acceptor antagonist to entrapment ligand H9 in vitro activeness and the function to human acceptor CX3-CR1].

AIM: To clarify the activeness of H9 in vitro and internalization and modulation of the surface chemokine receptor CX3CR1 induced by H9, To discuss the influence of H9 on the chemokine receptor CX3CR1. METHODS: Inhibition by chemotactic peptide on the physiological detection of chemokine induced cell migration activity. Flowcytometry examined the effection of H9 on intracellular calcium. Laser scanning confocal microscopy and flow cytometry were used to determine the quality and quantity of CX3CR1 internalization. RESULTS: H9 was able to block the migration induced by chemokine receptor. In the chemoattraction test, H9 was unable to induce the chemotactic movement, and it does not affect the signal transduction and activeness of cells. It was found that H9 could induce internalization with a maximal rate of 70%, at the concentration of 200 ng/mL. The internalized CX3CR1 molecules could recycled to the cell surface. CONCLUSION: H9 makes human CX3CR1 internalize. After internalizing, the CX3CR1 receptor recirculates the cell surface. It does not affect CX3CR1 physiology function. H9 could be used as a specificity anti-virus peptide.

[Acute toxicity of CCR5 antagonist C25P polypeptide in mice and its carcinogenicity in vitro].

OBJECTIVE: To study the acute toxicity of C25P polypeptide, a CCR5 antagonist, in mice and its carcinogenic effect in vitro. METHODS: The acute toxicity of C25P polypeptide in mice was assessed by determining the maximum tolerated dose (MTD). The mice were given C25P at the dose of 3.64 g/kg by tail vein injection, and the control mice received saline (40 ml/kg) injection. The mice were continuously observed for 14 days after the administration and sacrificed on day 14 for routine blood test, examination of the blood biochemistry and pathological examination. The carcinogenicity of C25P polypeptide in vitro was evaluated in cultured cell lines by chromosome aberration test, cell transformation test and non-anchorage dependent growth test. RESULTS: No mice died following administration of the drug, but 3 mice showed mild adverse reactions. The rats in both groups showed an increase in the body weight at a comparable rate. GPT increased and ALP decreased significantly in C25P polypeptide group (P<0.05). Most of the organs of the rats treated with in C25P polypeptide remained normal, but 3 mice showed pathologies in the lung, spleen and liver. Chromosome aberration test, cell transformation test and non-anchorage-dependent growth test all yielded negative results for C25P polypeptide. CONCLUSION: C25P polypeptide is a low-toxicity drug that produces no apparent acute toxicity in mice or obvious carcinogenicity in vitro.

A fungal enzyme with the ability of aflatoxin B1 conversion: purification and ESI-MS/MS identification.

Armillariella tabescens, a Chinese edible and medicinal fungus, whose multienzyme exist ability of AFB(1)-converting, and ADTZ (aflatoxin-detoxizyme) had previously purified from the A. tabescens multienzyme monitored by AFB(1) conversion(.) However, the enzyme now confirmed an oxidase and renamed aflatoxin-oxidase (AFO). In this paper, AFO was purified by an economical and practical three-step procedure monitored by AFB(1) conversion. And ESI-MS/MS analysis was done for identification of AFO. The following database searching (Protein Blast on NCBI) results did not show any homologous oxidase protein, which implied that AFO was mostly a new oxidase differing from other reported aflatoxin-converting enzymes such as fungal laccase and horse radish peroxidase. HPTLC analysis of the purified AFO activity suggested that the enzyme reacted at the bisfuran ring of AFB(1) which was the key toxic structure. Therefore, all these investigations implied a new choice for biodegradation of aflatoxin in foods and feeds with the practical application of AFO.

[SYBR green I real-time polymerase chain reaction for detection of Norovirus II in the shellfish].

We set up an SYBR Green I real-time RT-PCR method for the detection of genogroup II Norovirus, and this method's primers were encompassed the conservative region of Norovirus II. The limit of the detection was 10(2) copies. The standard curve's linear range was 10(2)-10(6) copies, correlation coefficient was 0.9952, the slope was -2.982, and the intercept was 35.84. This method possessed specificity for genogroup II Norovirus, without any cross-reaction with rotavirus, adenovirus, hepatitis A virus or astrovirus. The coefficients of variation (CV) of the C(t) values of the standard plasmid were 0.95%-1.69% (n = 5) in intra-assay and 0.87%-1.24% (n = 3) in inter-assay. We used this method to detect 30 shellfish samples, and found 3 samples were positive. This method is sensitive, specific and reliable for Norovirus II. It can be used to detect the Norovirus II in the shellfish rapidly.