51 research outputs found

    Relative Quantification of Protein-Protein Interactions Using a Dual Luciferase Reporter Pull-Down Assay System

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    The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins

    Stability of important antibodies for kidney disease: pre-analytic methodological considerations

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    Background The importance of circulating antibodies as biomarkers of kidney disease has recently been recognized. However, no study has systematically described the methodology of sample preparation and storage regarding antibodies as biomarkers of kidney disease. It remains unknown whether repetitive freeze-thaw cycles, physical disturbances, storage at different temperatures or for different periods of time, or haemolytic or turbid serum samples affect antibody measurements. The aim of this study was to investigate the stabilities of antibodies associated with kidney disease in serum samples under various relevant clinical and research conditions. Methods We stored serum samples in the following different conditions: repetitive freeze-thaw cycles (1, 6 or 12 times), long-term storage (7 or 12 months at −80 °C), physical disturbance (1 or 8 h), and storage at 4 °C (1, 3 or 6 weeks) and room temperature (1 or 7 days). The stabilities of the anti-phospholipase A2 receptor (anti-PLA2R), anti-glomerular basement membrane, anti-myeloperoxidase and anti-proteinase 3 antibodies were evaluated with enzyme-linked immunosorbent assays (ELISA). Results We found that repetitive freeze-thaw cycles did not have a significant effect on the stabilities of the abovementioned antibodies in clear serum samples. The ELISA readings of haemolytic and turbid serum samples tended to increase and decrease, respectively. Neither long-term storage at −80 °C nor physical disturbance had a significant effect on anti-PLA2R antibody stability in sealed serum samples. The concentrations of most of these antibodies increased in unsealed serum samples that were stored at 4 °C for more than 6 weeks or at room temperature for more than 7 days. Discussion Our findings revealed that the abovementioned circulating antibodies that are used as biomarkers for kidney disease had stable physicochemical properties, structures and immunoreactivities such that they were not influenced by repetitive freeze-thaw cycles, physical disturbances or long-term storage at −80 °C. However, the ELISA readings tended to change for haemolytic, turbid and unsealed serum samples

    Widely Targeted Metabolomics Revealed the Dynamic Changes of Metabolites during the Formation of Goose Fatty Liver

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    To understand the composition and dynamic changes of metabolites during the formation of goose fatty liver, the metabolite profiles of goose liver at three overfeeding stages were analyzed using widely targeted metabolomics. Three 70-day-old Landes geese with similar body conditions from the same batch were selected randomly for slaughter at the early (day 7), middle (day 16) and late (day 25) overfeeding stages, separately. The tip of the larger liver lobe was collected for widely targeted metabolomic analysis. The results showed that: (1) a total of 1 153 metabolites belonging to 19 classes including amino acids, organic acids, nucleotides and lipids were detected in the liver of geese at the three overfeeding stages; (2) principal component analysis (PCA) showed significant differences in the metabolic profiles of goose liver at the three stages, and identified 142 and 92 differential metabolites at the early versus middle stage, and the middle versus late stage, respectively, the major ones being amino acids and their derivatives, as well as organic acids and their derivatives; and (3) Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analysis indicated that the pathways involved in fatty acid biosynthesis, vitamin B6 metabolism, linoleic acid metabolism, lysine degradation, arginine biosynthesis, arachidonic acid metabolism and amino acid biosynthesis changed significantly during the formation of goose fatty liver. This study found that most of the differential metabolites were involved in fatty acid synthesis during goose fatty liver formation. Moreover, the contents of transport-related metabolites showed a continuous increasing trend. Findings in this study will not only enrich the theoretical knowledge of poultry liver metabolism, but also provide a theoretical basis for the precise nutritional regulation and efficient production of high-quality goose fatty liver

    Negatively Charged Composite Nanofibrous Hydrogel Membranes for High-Performance Protein Adsorption

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    Nanofibrous materials are considered as promising candidates for fabricating high-efficiency chromatography media, which are urgently needed in protein pharmaceuticals purification and biological research, yet still face several bottlenecks. Herein, novel negatively charged composite nanofibrous hydrogel membranes (NHMs) are obtained by a facile combination of electrospinning and surface coating modification. The resulting NHMs exhibit controllable morphologies and chemical structures. Benefitting from the combined effect of the stable framework of silicon dioxide (SiO2) nanofiber and the function layer of negatively charged hydrogel, as well as good pore connectivity among nanofibers, NHMs exhibit a high protein adsorption capacity of around 1000 mg g−1, and are superior to the commercial cellulose fibrous adsorbent (Sartobind®) and the reported nanofibrous membranous adsorbents. Moreover, due to their relatively stable physicochemical and mechanical properties, NHMs possess comprehensive adsorption performance, favorable resistance to acid and solvents, good selectivity, and excellent regenerability. The designed NHMs composite adsorbents are expected to supply a new protein chromatography platform for effective protein purification in biopharmaceuticals and biochemical reagents

    Developing Village-Based Green Economy in an Endogenous Way: A Case Study from China

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    The idea of green economy is being taken seriously all over the world. For developing countries, the key to developing green economy is to strike a balance between environmental protection and economic development. As the largest developing country, China has been exploring scientific schemes to deal with the relationship between environmental protection and economic development. Developing rural tourism is an important way to transform ecological advantages into economic benefits. However, the role of rural tourism remains controversial. No scholars have yet provided solutions for village-level practices in developing countries from the perspective of endogenous development theory. Taking Yucun, a village in Zhejiang Province as an example, this paper reveals the endogenous way of green economy development at the village level through the method of case study. It is confirmed that the key to transforming rural ecological advantages into economic benefits is to cultivate the village’s endogenous development capacity, including activating local resources, cultivating local identity, stimulating local participation, and building a collaborative network. Only by implementing the endogenous development mode in rural areas cannot only stimulate the positive role of rural tourism and form a virtuous cycle, but also avoid the negative effects of rural tourism previously pointed out by scholars

    Establishment of a rat model for uterine leiomyomas based on Western and traditional Chinese medicine theories

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    Uterine leiomyomas (ULs) are benign monoclonal tumors that arise from the underlying myometrial tissue in the uterus. Effective therapies are still lacking because of poor understanding of the pathophysiology and epidemiology. Hence, it is urgent to establish efficient animal models to screen novel anti-UL therapies. In this study, for the first time, traditional Chinese medicine and Western medicine were combined to establish an animal model of ULs in rats. In order to evaluate the function and value of the novel model, it was compared with other models. The long-term and short-term rat models for ULs were established using progesterone and diethylstilbestrol. Rats in Qi stagnation and blood stasis group were injected with epinephrine hydrochloride and received chronic unpredictable stress for two weeks. Rats in combining disease with syndrome group (CDWSG) received not only epinephrine hydrochloride injection and chronic unpredictable stress but also progesterone and diethylstilbestrol treatment. We analyzed differences in organ coefficient, uterus size, uterine pathology, concentrations of progesterone, estradiol, progesterone receptor, estrogen receptor, expression of desmin, α-smooth muscle actin, and vimentin among the five groups. The animal model of ULs was successfully constructed by loading the rats with estrogen and progesterone. The rat model of CDWSG was more stable than other groups and the method was the most efficient
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