Phase transition of a symmetric diblock copolymer induced by nanorods with different surface chemistry.

We investigate the phase transition of a symmetric diblock copolymer induced by nanorods with different surface chemistry. The results demonstrate that the system occurs the phase transition from a disordered structure to ordered parallel lamellae and then to the tilted layered structure as the number of rods increases. The dynamic evolution of the domain size and the order parameter of the microstructure are also examined. Furthermore, the influence of rod property, rod-phase interaction, rod-rod interaction, rod length, and polymerization degree on the behavior of the polymer system is also investigated systematically. Moreover, longer amphiphilic nanorods tend to make the polymer system form the hexagonal structure. It transforms into a perpendicular lamellar structure as the polymerization degree increases. Our simulations provide an efficient method for determining how to obtain the ordered structure on the nanometer scales and design the functional materials with optical, electronic, and magnetic properties.

Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris.

Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.

Resveratrol analogue 3,4,4'-trihydroxy-trans-stilbene induces apoptosis and autophagy in human non-small-cell lung cancer cells in vitro.

AIM: To investigate the effects of 3,4,4'-trihydroxy-trans-stilbene (3,4,4'-THS), an analogue of resveratrol, on human non-small-cell lung cancer (NSCLC) cells in vitro. METHODS: Cell viability of NSCLC A549 cells was determined by MTT assay. Cell apoptosis was evaluated using flow cytometry and TUNEL assay. Cell necrosis was evaluated with LDH assay. The expression of apoptosis- or autophagy-associated proteins was measured using Western blotting. The formation of acidic compartments was detected using AO staining, neutral red staining and Lysotracker-Red staining. LC3 punctae were analyzed with fluorescence microscopy. RESULTS: Treatment with 3,4,4'-THS (10-80 µmol/L) concentration-dependently inhibited the cell viability. It did not cause cell necrosis, but induced apoptosis accompanied by up-regulation of cleavaged PARP, caspase3/9 and Bax, and by down-regulation of Bcl-2 and surviving. It also increased the formation of acidic compartments, LC3-II accumulation and GFP-LC3 labeled autophagosomes in the cells. It inhibited the mTOR-dependent pathway, but did not impair autophagic flux. 3,4,4'-THS-induced cell death was enhanced by the autophagy inhibitors 3-MA (5 mmol/L) or Wortmannin (2 µmol/L). Moreover, 3,4,4'-THS treatment elevated the ROS levels in the cells, and co-treatment with 3-MA further elevated the ROS levels. 3,4,4'-THS-induced apoptosis and autophagy in the cells was attenuated by NAC (10 mmol/L)Conclusion:3,4,4'-THS induces both apoptosis and autophagy in NSCLC A549 cells in vitro. Autophagy inhibitors promote 3,4,4'-THS-induced apoptosis of A549 cells, thus combination of 3,4,4'-THS and autophagy inhibitor provides a promising strategy for NSCLC treatment.

Identification of important residues of insulin-like peptide 5 and its receptor RXFP4 for ligand-receptor interactions.

Insulin-like peptide 5 (INSL5) is an insulin/relaxin superfamily peptide involved in the regulation of glucose homeostasis by activating its receptor RXFP4, which can also be activated by relaxin-3 in vitro. To determine the interaction mechanism of INSL5 with its receptor RXFP4, we studied their electrostatic interactions using a charge-exchange mutagenesis approach. First, we identified three negatively charged extracellular residues (Glu100, Asp104 and Glu182) in human RXFP4 that were important for receptor activation by wild-type INSL5. Second, we demonstrated that two positively charged B-chain Arg residues (B13Arg and B23Arg) in human INSL5 were involved in receptor binding and activation. Third, we proposed probable electrostatic interactions between INSL5 and RXFP4: the B-chain central B13Arg of INSL5 interacts with both Asp104 and Glu182 of RXFP4, meanwhile the B-chain C-terminal B23Arg of INSL5 interacts with both Glu100 and Asp104 of RXFP4. The present electrostatic interactions between INSL5 and RXFP4 were similar to our previously identified interactions between relaxin-3 and RXFP4, but had subtle differences that might be caused by the different B-chain C-terminal conformations of relaxin-3 and INSL5 because a dipeptide exchange at the B-chain C-terminus significantly decreased the activity of INSL5 and relaxin-3 to receptor RXFP4.

The highly conserved negatively charged Glu141 and Asp145 of the G-protein-coupled receptor RXFP3 interact with the highly conserved positively charged arginine residues of relaxin-3.

Relaxin-3 is a newly identified insulin/relaxin superfamily peptide that plays a putative role in the regulation of food intake and stress response by activating its cognate G-protein-coupled receptor RXFP3. Relaxin-3 has three highly conserved arginine residues, B12Arg, B16Arg and B26Arg. We speculated that these positively charged arginines may interact with certain negatively charged residues of RXFP3. To test this hypothesis, we first replaced the negatively charged residues in the extracellular domain of RXFP3 with arginine, respectively. Receptor activation assays showed that arginine replacement of Glu141 or Asp145, especially Glu141, significantly decreased the sensitivity of RXFP3 to wild-type relaxin-3. In contrast, arginine replacement of other negatively charged extracellular residues had little effect. Thus, we deduced that Glu141 and Asp145, locating at the extracellular end of the second transmembrane domain, played a critical role in the interaction of RXFP3 with relaxin-3. To identify the ligand residues interacting with the negatively charged EXXXD motif of RXFP3, we replaced the three conserved arginines of relaxin-3 with negatively charged glutamate or aspartate, respectively. The mutant relaxin-3s retained the native structure, but their binding and activation potencies towards wild-type RXFP3 were decreased significantly. The compensatory effects of the mutant relaxin-3s towards mutant RXFP3s suggested two probable interaction pairs during ligand-receptor interaction: Glu141 of RXFP3 interacted with B26Arg of relaxin-3, meanwhile Asp145 of RXFP3 interacted with both B12Arg and B16Arg of relaxin-3. Based on these results, we proposed a relaxin-3/RXFP3 interaction model that shed new light on the interaction mechanism of the relaxin family peptides with their receptors.

[Effect of modified zuoguiwan on Th17/Treg subpopulation of estrogen deficiency induced bone loss mice].

OBJECTIVE: To observe the effect of Modified Zuoguiwan (MZ) on the balance between helper T cell subsets 17 (Th17) and regulatory T cell subsets (Treg) in estrogen deficiency induced bone loss mice and to explore its mechanism. METHODS: Totally 50 BALB/c mice were divided into the sham-operation group, the ovariectomy model group, the low dose MZ group, the middle dose MZ group, and the high dose MZ group by random digit table, 10 in each group. Mice in the low, middle, and high dose MZ groups were respectively administered with MZ at the daily dose of 7.25, 14.50, and 29.00 g/kg by gastrogavage, 0.5 mL each time for 12 successive weeks. Meanwhile, mice in the sham-operation group and the ovariectomy model group were administered with equal volume by gastrogavage, 0.50 mL each time. The serum estradiol (E2) level was assessed by enzyme linked immunosorbent assay (ELISA). Bone mineral density (BMD) of thigh bone was measured with dual energy X ray absorptiometry. In addition, the population of Th17/Treg subsets in spleen mononuclear cells was analyzed by extracellular and intracellular staining method using flow cytometry. Moreover, the mRNA expression of IL-17A and TGF-ß in the spleen mononuclear cells was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Compared with the sham-operation group, both E2 and BMD significantly decreased, the percentage of Th17 subset and Th17/Treg ratio both increased, the percentage of Treg subset obviously decreased, the expression of IL-17A mRNA significantly increased, and the expression of TGF-ß mRNA significantly decreased in the ovariectomy model group (all P < 0.05). Compared with the model group, BMD obviously increased, the percentage of Th17 subset and Th17/Treg ratio both decreased, the percentage of Treg subset obviously increased, the expression of IL-17A mRNA significantly decreased, and the expression of TGF-ß mRNA significantly increased in the middle dose MZ group and the high dose MZ group (all P < 0. 05). Correlation analyses showed that BMD was positively related to both the serum E2 level and the percentage of Treg subset (P < 0.05), but negatively related to the percentage of Th17 subset (P < 0.05). In addition, the serum E2 level was positively related to the percentage of Treg subset, but obviously negatively related to that of Th17 subset (P < 0.05). CONCLUSIONS: There was correlation between Th17/Treg imbalance and E2 deficient bone loss. MZ could decrease the proportion of Th17 subset, but elevate the proportion of Treg subset in E2 deficient bone loss mice. It could achieve therapeutic effect through adjusting the balance of Th17/Treg in E2 deficient bone loss mice.

Preoperative contrast-enhanced computed tomography-based radiomics model for overall survival prediction in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy with a rising incidence worldwide. The prognosis of HCC patients after radical resection remains poor. Radiomics is a novel machine learning method that extracts quantitative features from medical images and provides predictive information of cancer, which can assist with cancer diagnosis, therapeutic decision-making and prognosis improvement. AIM: To develop and validate a contrast-enhanced computed tomography-based radiomics model for predicting the overall survival (OS) of HCC patients after radical hepatectomy. METHODS: A total of 150 HCC patients were randomly divided into a training cohort (n = 107) and a validation cohort (n = 43). Radiomics features were extracted from the entire tumour lesion. The least absolute shrinkage and selection operator algorithm was applied for the selection of radiomics features and the construction of the radiomics signature. Univariate and multivariate Cox regression analyses were used to identify the independent prognostic factors and develop the predictive nomogram, incorporating clinicopathological characteristics and the radiomics signature. The accuracy of the nomogram was assessed with the concordance index, receiver operating characteristic (ROC) curve and calibration curve. The clinical utility was evaluated by decision curve analysis (DCA). Kaplan-Meier methodology was used to compare the survival between the low- and high-risk subgroups. RESULTS: In total, seven radiomics features were selected to construct the radiomics signature. According to the results of univariate and multivariate Cox regression analyses, alpha-fetoprotein (AFP), neutrophil-to-lymphocyte ratio (NLR) and radiomics signature were included to build the nomogram. The C-indices of the nomogram in the training and validation cohorts were 0.736 and 0.774, respectively. ROC curve analysis for predicting 1-, 3-, and 5-year OS confirmed satisfactory accuracy [training cohort, area under the curve (AUC) = 0.850, 0.791 and 0.823, respectively; validation cohort, AUC = 0.905, 0.884 and 0.911, respectively]. The calibration curve analysis indicated a good agreement between the nomogram-prediction and actual survival. DCA curves suggested that the nomogram had more benefit than traditional staging system models. Kaplan-Meier survival analysis indicated that patients in the low-risk group had longer OS and disease-free survival (all P < 0.0001). CONCLUSION: The nomogram containing the radiomics signature, NLR and AFP is a reliable tool for predicting the OS of HCC patients.

Dual Enzyme-Like Performances of PLGA Grafted Maghemite Nanocrystals and Their Synergistic Chemo/Chemodynamic Treatment for Human Lung Adenocarcinoma A549 Cells.

In recent years, the emergence of non-toxic but catalytically active inorganic nanoparticles has attracted great attention for cancer treatment, but the therapeutic effect has been affected by the limited reactive oxygen species in tumors. Therefore, the combination of chemotherapy and chemodynamic therapy is regarded as a promising therapeutic strategy. In this paper, we reported the preparation and bioactivity evaluation of poly(lactic acid-co-glycolic acid) (PLGA) grafted-γ-Fe2O3 nanoparticles with dual response of endogenous peroxidase and catalase like activities. Our hypothesis is that PLGAgrafted γ-Fe2O3 nanoparticles could be used as a drug delivery system for the anti-tumor drug doxorubicin to inhibit the growth of lung adenocarcinoma A549 cells; meanwhile, based on its mimic enzyme properties, this kind of nanoparticles could be combined with doxorubicin in the treatment of A549 cells. Our experimental results showed that the PLGAgrafted γ-Fe2O3 nanoparticles could simulate the activity of catalase and decompose hydrogen peroxide into H2O and oxygen in neutral tumor microenvironment, thus reducing the oxidative damage caused by hydrogenperoxide to lung adenocarcinoma A549 cells. In acidic microenvironment, PLGA grafted γ-Fe2O3 nanoparticles could simulate the activity of peroxidase and effectively catalyze the decomposition of hydrogen peroxide to generate highly toxic hydroxyl radicals, which could cause the death of A549 cells. Furthermore, the synergistic effect of peroxidase-like activity of PLGA-grafted γ-Fe2O3 nanoparticles and doxorubicin could accelerate the apoptosisand destruction of A549 cells, thus enhancing the antitumor effect of doxorubicin-loaded PLGA-grafted γ-Fe2O3 nanoparticles. Therefore, this study provides an effective nanoplatform based on dual inorganic biomimetic nanozymes for the treatment of lung cancer.

[Effects of siltuximab on the interleukin-6/Stat3 signaling pathway in ovarian cancer].

OBJECTIVE: To study the effects of siltuximab on the interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (Stat3) signaling pathway in ovarian epithelial carcinoma. METHODS: (1) Expressions of IL-6 in ovarian cancer patient specimens were assessed by immunohistochemistry. (2) Expression of phosphorylation Stat3 (pStat3) protein in siltuximab and IL-6 treated SKOV3 cell lines was determined by western blot, and expression levels of Stat3-induced bcl-XL, MCL-1, survivin, in siltuximab treated SKOV3/TR and CAOV3/TR cells lines were also determined by western blot. (3) Real-time image analysis was used to study the nuclear translocation of pEGFP-Stat3 fusion protein in ovarian cancer cell line SKOV3-pEGFP-Stat3 treated with siltuximab and IL-6. (4) Paclitaxel sensitivity in siltuximab treated SKOV3/TR and CAOV3/TR cell lines were assessed using the methyl thiazolyl tetrazolium (MTT). The 50% inhibiting concentration (IC(50)) was defined as the paclitaxel concentration required to decrease the A(490) value to 50%. RESULTS: (1) There were significantly difference in IL-6 staining density and the positive rate of IL-6 protein stained among the metastatic, and drug-resistant recurrent tumors, and matched primary tumors [69% (18/26)] vs. 77% (20/26) vs. 23% (6/26), P < 0.05]. (2)A clear increase in Stat3 phosphorylation levels was observed in the IL-6-treated SKOV3 cell lines as compared to the SKOV3 cell lines. When the IL-6-treated SKOV3 cells were incubated with siltuximab with a range of concentrations of 0.001, 0.01, 0.1, 1.0 and 10 µg/ml, there were trends toward reduced pStat3 expression in the treated cell lines. Compared without treatment with siltuximab, the expression of the anti-apoptotic proteins MCL-1, bcl-XL and survivin in SKOV3/TR and CAOV3/TR cell lines were significantly decreased after treated with siltuximab. (3) In resting cells, the majority of pEGFP-Stat3 was cytoplasmic until the addition of human IL-6, which promptly induced the translocation of fluorescent Stat3 molecules to the nucleus. Exposure of cells to siltuximab with a range of concentrations of 0.001, 0.01, 0.1, 1.0 and 10 µg/ml, followed by an incubation in IL-6 significantly reduced pEGFP-Stat3 nucleocytoplasmic translocation. (4) MTT cytotoxicity assay demonstrated that siltuximab increased paclitaxel-induced cell death and partially overcame paclitaxel resistance. Treated with siltuximab (1 and 10 µg/ml), the paclitaxel IC(50) value of siltuximab in SKOV3/TR (0.49, 0.19 µg/ml) and CAOV3/TR (0.0010, 0.0008 µg/ml) cells were significantly lower than those in untreated cells (0.71, 0.0021 µg/ml; all P < 0.05). CONCLUSIONS: These results demonstrated that siltuximab effectively block the IL-6 signaling pathways, which. Blockage of IL-6 signaling may provide benefits for the treatment of ovarian cancer.

Gene expression of halophyte Kosteletzkya virginica seedlings under salt stress at early stage.

Gene differential expression of Kosteletzkya virginica seedlings under salt stress at two time points (2, 24 h) in roots and leaves was analyzed using the cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. Polymorphic transcript-derived fragments (TDFs) among control plants and salt-treated plants were grouped into four main differential expression patterns: repression (A), de novo induction (B), up-regulation (C) and down-regulation (D). Among them, 34 differentially expressed gene fragments were homologous to known genes from other species and 4 were sequences with unknown functions. These differentially expressed genes can be classified into four groups according to their putative functions: (1) genes for re-establishing ion homeostasis and protecting the plant from stress damage; (2) genes involved in metabolism or energy and resuming plant growth and development under salt stress; (3) genes involved in regulation of gene expression; (4) genes for signal transduction. Changes of eight differentially expressed genes were confirmed by quantitative real time RT-PCR.

[Analysis of gene expression involved in the response to salt stress in the dicot halophyte Kosteletzkya virginica L. seedlings].

Kosteletzkya virginica L. Presl. is an obligate wetland species indigenous to southeastern US. Its niche in salt marsh foretells its high salinity tolerance. cDNA-AFLP technique was used to identify the gene transcriptional profiles of leaves and roots from K. virginica seedlings under salt stress in order to clarify the molecular architecture of stress tolerance in the dicot halophyte. Expression analysis over time intervals and under various salt stresses in leaves or roots showed that the quantitatively expressed pattern (in which genes were quantitatively up- or down-regulated under salt stress or fluctuate with different NaCl concentrations) was more prevalent than the qualitatively expressed pattern (in which genes were induced or silenced under salt stress) in K. virginica seedlings under salt stress. The qualitative pattern was appreciably more predominant than the quantitative one only in roots when exposed to salt stress for 2 h. Although each expression pattern was observed in leaves as well as in roots, the percentage of genes (i.e., up-/down-regulated or induced/silenced under salt stress) was dynamically changeable under salt stress at different time intervals. All these results indicated that there was no established formula of gene expression patterns in deciphering the sophisticated mechanism of plant salinity tolerance, considering that plants undergo a series of dynamically physiological and metabolic pathways in sensing and response to salt stress for different tissues and during different stages of stress. A number of Trivially distributed file system (TDFs) up-regulated or induced under salt stress from leaves and roots were sequenced, and the sequences were blasted against the NCBI non-redundant protein database using translated nucleotide query (Blastx). The TDFs from K. virginica seedlings involved in sensing and response to salt stress can be classified at least into three groups according to their putative functions: (1) genes for re-establishing ionic homeostasis or preventing from damage (specially genes for transporter); (2) genes for resuming plant growth and development under salt stress, such as key enzymes involved in energy synthesis or hormone regulatory pathway; (3) genes for signal transduction and so on. The relationship of expression patterns of these TDFs with the molecular mechanism of salt tolerance in K. virginica was discussed.

[Effects of yougui pill on phenotype change of thymic dendritic cells induced by glucocorticoid in mice].

OBJECTIVE: To investigate the influence of glucocorticoid on phenotype of thymic dendritic cells in mice and to investigate the protective effect of Yougui Pill (YGP) on it. METHODS: BALB/c mice allocated in the group A and B were treated respectively with 10 mg/kg hydrocortisone, alone and combined with 20.81 g/kg YGP. The control mice were treated with normal saline. The changes before and after treatment of I-A(d) and H-2K(d) antigen presentation molecules expression in CD11c(+) and CD45(+) thymic dendritic cells of mice were analyzed by flow cytometry assay, and the expression of intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1 (LFA-1) mRNA in thymocytes were determined by RT-PCR as well. RESULTS: The percentage of I-A(d+) and H-2K(d+) in CD11c(+) in Group A after treatment was 46.77 +/- 4.32% and 64.34 +/- 7.69% respectively, as compared with those in the control group (65.81 +/- 7.69% and 31.88 +/- 5.01%), the percentage of I-A(d+) was lower and that of H-2K(d+) was higher significantly (all P <0.01). Meantime, the expression of ICAM-1 and LFA-1 in thymocyte in Group A (30.11 +/- 2.51% and 30.40 +/- 3.77%) was significantly lower than that in the control group (46.35 +/- 3.34% and 47.28 +/- 2.91%) respectively (P <0.01). Changes in Group B showed that treated by hydrocortisone in combination with YGP, the above-mentioned hydrocortisone-induced changes could be obviously reversed, the outcome of CD11c(+) I-A(d+) was 54.19 +/- 5.08%, ICAM-1 33.97 +/- 2.04% and LFA-1 34.80 +/- 2.92%, the difference between the two treated groups in these indexes all showed statistical significance (P <0.05). CONCLUSION: Glucocorticoidcan inhibit the expression of major histocompatibility complex class II antigen molecule, but promote the expression of major histocompatibility complex class I in CD11c(+) and CD45(+) dendritic cells, down-regulate ICAM-1 and LFA-1 transcription, while the tonifying yang recipe, YGP, has a dominant protective effect against the above actions of glucocorticoid.

[Expression of multiepitope antigenic gene of Toxoplasma gondii in transgenic tomatoes].

OBJECTIVE: To obtain the transgenic tomato plant expressed multiepitope antigenic gene of Toxoplasma gondii (Tg-MAG). METHODS: Tg-MAG under the control of E35S promoter, E35S-E81.1 chimeric promoter and E8 2.2 promoter in the plant expression vectors was transferred into the tomato cotyledons and hypocotyls via Agrobacterium-mediated T-DNA transformation. The candidate buds were obtained and elongated by kanamycin resistant screening. After further rooting selection, the selected tomato lines were trained and transplanted to soil and grown in a greenhouse till blossoming and bearing fruit. The transgenic tomato plants were identified using PCR, RT-PCR and Western blotting. RESULTS: Tg-MAG was introduced into the transgenic plant genomic DNA and transcribed correctly at expected size 360 bp identified by PCR and RT-PCR. Western blotting results indicated that the recombinant Tg-MAG from 8 transgenic lines, expressed in tomato fruits at expected size 11900, 2 of them showed strong reaction band with HRP labeled McAb K7H3 against the major surface antigen 1( SAG1) of T. gondii tachyzoites. CONCLUSION: Tg-MAG transgenic tomato lines were obtained. Tg-MAG recombinant protein with good immune activity was expressed successfully in transgenic tomato fruits.

Mechanism for insulin-like peptide 5 distinguishing the homologous relaxin family peptide receptor 3 and 4.

The relaxin family peptides play a variety of biological functions by activating four G protein-coupled receptors, RXFP1-4. Among them, insulin-like peptide 5 (INSL5) and relaxin-3 share the highest sequence homology, but they have distinct receptor preference: INSL5 can activate RXFP4 only, while relaxin-3 can activate RXFP3, RXFP4, and RXFP1. Previous studies suggest that the A-chain is responsible for their different selectivity for RXFP1. However, the mechanism by which INSL5 distinguishes the homologous RXFP4 and RXFP3 remains unknown. In the present work, we chemically evolved INSL5 in vitro to a strong agonist of both RXFP4 and RXFP3 through replacement of its five B-chain residues with the corresponding residues of relaxin-3. We identified four determinants (B2Glu, B9Leu, B17Tyr, and a rigid B-chain C-terminus) on INSL5 that are responsible for its inactivity at RXFP3. In reverse experiments, we grafted these determinants onto a chimeric R3/I5 peptide, which contains the B-chain of relaxin-3 and the A-chain of INSL5, and retains full activation potency at RXFP3 and RXFP4. All resultant R3/I5 mutants retained high activation potency towards RXFP4, but most displayed significantly decreased or even abolished activation potency towards RXFP3, confirming the role of these four INSL5 determinants in distinguishing RXFP4 from RXFP3.

Nanoluciferase as a novel quantitative protein fusion tag: Application for overexpression and bioluminescent receptor-binding assays of human leukemia inhibitory factor.

Nanoluciferase (NanoLuc) is a newly developed small luciferase reporter with the brightest bioluminescence reported to date. In the present work, we developed NanoLuc as a novel quantitative protein fusion tag for efficient overexpression in Escherichia coli and ultrasensitive bioluminescent assays using human leukemia inhibitory factor (LIF) as a model protein. LIF is an interleukin 6 family cytokine that elicits pleiotropic effects on a diverse range of cells by activating a heterodimeric LIFR/gp130 receptor. Recombinant preparation of the biologically active LIF protein is quite difficult due to its hydrophobic nature and three disulfide bonds. Using the novel NanoLuc-fusion approach, soluble 6×His-NanoLuc-LIF fusion protein was efficiently overexpressed in E. coli and enzymatically converted to monomeric mature LIF. Both the mature LIF and the NanoLuc-fused LIF had high biological activities in a leukemia M1 cell proliferation inhibition assay and in a STAT3 signaling activation assay. The NanoLuc-fused LIF retained high binding affinities with the overexpressed LIFR (Kd = 1.4 ± 0.4 nM, n = 3), the overexpressed LIFR/gp130 (Kd = 115 ± 8 pM, n = 3), and the endogenously expressed LIFR/gp130 (Kd = 33.1 ± 3.2 pM, n = 3), with a detection limit of less than 10 receptors per cell. Thus, the novel NanoLuc-fusion strategy not only provided an efficient approach for preparation of recombinant LIF protein but also provided a novel ultrasensitive bioluminescent tracer for ligand-receptor interaction studies. The novel NanoLuc-fusion approach could be extended to other proteins for both efficient sample preparation and various bioluminescent quantitative assays in future studies.

The electrostatic interactions of relaxin-3 with receptor RXFP4 and the influence of its B-chain C-terminal conformation.

Relaxin-3 (also known as insulin-like peptide 7) is an insulin/relaxin-superfamily peptide hormone that can bind and activate three relaxin-family peptide receptors: RXFP3, RXFP4, and RXFP1. Recently, we identified key electrostatic interactions between relaxin-3 and its cognate receptor RXFP3 by using a charge-exchange mutagenesis approach. In the present study, the electrostatic interactions between relaxin-3 and RXFP4 were investigated with the same approach. Mutagenesis of the negatively charged extracellular residues of human RXFP4 identified a conserved EXXXD(100-104) motif that is essential for RXFP4 activation by relaxin-3. Mutagenesis of the conserved positively charged Arg residues of relaxin-3 demonstrated that B12Arg, B16Arg and B26Arg were all involved in the binding and activation of RXFP4, especially B26Arg. The activity complementation between the mutant ligands and the mutant receptors suggested two probable electrostatic interaction pairs: Glu100 of RXFP4 versus B26Arg of relaxin-3, and Asp104 of RXFP4 versus both B12Arg and B16Arg of relaxin-3. For interaction with the essential EXXXD motifs of both RXFP3 and RXFP4, a folding-back conformation of the relaxin-3 B-chain C-terminus seems to be critical, because it brings B26Arg sufficiently close to B12Arg and B16Arg. To test this hypothesis, we replaced the conserved B23Gly-B24Gly dipeptide of relaxin-3 with an Ala-Ser dipeptide that occupied the corresponding position of insulin-like peptide 5 and resulted in an extended helical conformation. The mutant relaxin-3 showed a significant decrease in receptor-activation potency towards both RXFP3 and RXFP4, suggesting that a folding-back conformation of the B-chain C-terminus was important for relaxin-3 to efficiently interact with the EXXXD motifs of both receptors.

Enhanced decolorization of azo dye in a small pilot-scale anaerobic baffled reactor coupled with biocatalyzed electrolysis system (ABR-BES): a design suitable for scaling-up.

A four-compartment anaerobic baffled reactor (ABR) incorporated with membrane-less biocatalyzed electrolysis system (BES) was tested for the treatment of azo dye (alizarin yellow R, AYR) wastewater (AYR, 200 mg L(-1); glucose, 1000 mg L(-1)). The ABR-BES was operated without and with external power supply to examine AYR reduction process and reductive intermediates with different external voltages (0.3, 0.5 and 0.7 V) and hydraulic retention times (HRT: 8, 6 and 4h). The decolorization efficiency in the ABR-BES (8h HRT, 0.5 V) was higher than that in ABR-BES without electrolysis, i.e. 95.1 ± 1.5% versus 86.9 ± 6.3%. Incorporation of BES with ABR accelerated the consumption of VFAs (mainly acetate) and attenuated biogas (methane) production. Higher power supply (0.7 V) enhanced AYR decolorization efficiency (96.4 ± 1.8%), VFAs removal, and current density (24.1 Am(-3) TCV). Shorter HRT increased volumetric AYR decolorization rates, but decreased AYR decolorization efficiency.

A membrane-free, continuously feeding, single chamber up-flow biocatalyzed electrolysis reactor for nitrobenzene reduction.

A new bioelectrochemical system (BES), a membrane-free, continuous feeding up-flow biocatalyzed electrolysis reactor (UBER) was developed to reduce oxidative toxic chemicals to less- or non-toxic reduced form in cathode zone with oxidation of electron donor in anode zone. Influent was fed from the bottom of UBER and passed through cathode zone and then anode zone. External power source (0.5 V) was provided between anode and cathode to enhance electrochemical reactions. Granular graphite and carbon brush were used as cathode and anode, respectively. This system was tested for the reduction of nitrobenzene (NB) using acetate as electron donor and carbon source. The influent contained NB (50-200 mg L(-1)) and acetate (1000 mg L(-1)). NB was removed by up to 98% mainly in cathode zone. The anode potential maintained under -480 mV. The maximum NB removal rate was up to 3.5 mol m(-3) TV d(-1) (TV=total empty volume) and the maximum aniline (AN) formation rate was 3.06 mol m(-3) TV d(-1). Additional energy required was less than 0.075 kWh mol(-1)NB. The molar ratio of NB removed vs acetate consumed varied from 4.3 ± 0.4 to 2.3 ± 0.1 mol mol(-1). Higher influent phosphate or acetate concentration helped NB removal rate. NB could be efficiently reduced to AN as the power supplied of 0.3 V.

Azo dye removal in a membrane-free up-flow biocatalyzed electrolysis reactor coupled with an aerobic bio-contact oxidation reactor.

Azo dyes that consist of a large quantity of dye wastewater are toxic and persistent to biodegradation, while they should be removed before being discharged to water body. In this study, Alizarin Yellow R (AYR) as a model azo dye was decolorized in a combined bio-system of membrane-free, continuous up-flow bio-catalyzed electrolysis reactor (UBER) and subsequent aerobic bio-contact oxidation reactor (ABOR). With the supply of external power source 0.5 V in the UBER, AYR decolorization efficiency increased up to 94.8±1.5%. Products formation efficiencies of p-phenylenediamine (PPD) and 5-aminosalicylic acid (5-ASA) were above 90% and 60%, respectively. Electron recovery efficiency based on AYR removal in cathode zone was nearly 100% at HRTs longer than 6 h. Relatively high concentration of AYR accumulated at higher AYR loading rates (>780 gm(-3) d(-1)) likely inhibited acetate oxidation of anode-respiring bacteria on the anode, which decreased current density in the UBER; optimal AYR loading rate for the UBER was 680 gm(-3) d(-1) (HRT 2.5 h). The subsequent ABOR further improved effluent quality. Overall the Chroma decreased from 320 times to 80 times in the combined bio-system to meet the textile wastewater discharge standard II in China.

[Establishment of the genotype-specific targets of botulinum neurotoxins types B and E based on reverse genetics].

OBJECTIVE: To establish the genotype-specific targets plasmids and engineered E.coli strains of botulinum neurotoxins (BoNT) types B and E based on reverse genetics. METHODS: The gene sequences of BoNT were obtained from GenBank and analyzed using DNAMAN, Lasergene, Vector NTI and BLAST. Two target fragments of BoNT/B and BoNT/E were anchored and then synthesized as 5 and 10 short DNA single strands, respectively. The full-length target sequences were amplified by overlapping PCR and subcloned into pMD 18-T vector, and the recombinant plasmids were identified by restriction enzyme digestion and sequencing. RESULTS: Sixty full-length sequences of 4 types of BoNT, namely types A, B, E, and F, were available in GenBank. Two target fragments, BoNT/B of 215 bp and BoNT/E of 360 bp, and their specific primer pairs were anchored after sequence analysis. pMD 18-T-BoNT/B and pMD 18-T-BoNT/E containing these two target sequences were confirmed. CONCLUSION: The engineered plasmids and E.coli stains containing the genotype-specific target fragments of BoNT/B and BoNT/E have been constructed successfully.