SummaryExosomes are membranous nanovesicles of endocytic origin that carry and transfer regulatory bioactive molecules and mediate intercellular communication between cells and tissues. Although seminal exosomes have been identified in human seminal plasma, their exact composition and possible physiologic function remain unknown. The objective of this study was to perform a comprehensive proteomics analysis of exosomes derived from human seminal plasma. Seminal exosomes were isolated and purified from 12 healthy donors using a 30% sucrose cushion‐based exosome‐isolation protocol, followed by characterization by western blot, transmission electron microscopy, and nanoparticle tracking analysis before performing extensive liquid chromatography tandem mass spectrometry proteomics analysis. The identified proteins were analyzed by bioinformatics analysis, and seminal exosomes‐associated proteins were selectively validated by western blot. A total of 1474 proteins were identified in all seminal exosomes samples, with Gene Ontology analysis demonstrating that these identified seminal exosomes‐associated proteins were mostly linked to ‘exosomes,’ ‘cytoplasm,’ and ‘cytosol.’ Bioinformatics analysis indicated that these proteins were mainly involved in biologic processes, including metabolism, energy pathways, protein metabolism, cell growth and maintenance, and transport. Of these identified proteins, PHGDH, LGALS3BP, SEMG1, ACTB, GAPDH, and the exosomal‐marker protein ALIX were validated by western blot. This study provided a more comprehensive description of the seminal exosomes proteome and could also be a resource for further screening of biomarkers and comparative proteomics studies, including those associated with male infertility and prostate cancer.

Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors are CL/RAMP2 (AM 1 receptor) or CL/RAMP3 (AM 2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY 1 , AMY 2 , and AMY 3 , respectively). How RAMPs change G protein-coupled receptor pharmacology is not fully understood. We exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering receptor pharmacology. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. We hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM receptors. Using site-directed mutagenesis, we introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-negative form of the calcitonin receptor [CT (a) ]. Agonist-stimulated cAMP production and cellsurface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was critical for determining AM potency and affinity, and Phe93 in RAMP1 was an important contributor to ␣CGRP potency at CGRP receptors. Mutant RAMP/CT (a) receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cellsurface expression of these complexes. This differs from the effect on CL-based receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacology.The calcitonin peptide family, in humans, comprises calcitonin, amylin (Amy), two forms of calcitonin gene-related peptide (CGRP; ␣ and ␤) and adrenomedullin (AM). A second version of AM, a 47-amino acid peptide named intermedin (IMD) or AM2, has been identified more recently (Fig. 1A) (Roh et al., 2004;Takei et al., 2004). Calcitonin-peptide family receptors are composed of G protein-coupled receptors and receptor activity-modifying proteins (RAMPs) (Poyner et al., 2002). For example, AM receptors are heteromers of the calcitonin receptor-like receptor (CL) with RAMP2 (AM 1 receptors) or RAMP3 (AM 2 receptors). CGRP 1 receptors are formed from CL/RAMP1 complexes. In contrast, Amy receptors comprise the calcitonin receptor with individual RAMPs to generate AMY 1 , AMY 2 , and AMY 3 receptor subtypes.Pharmacological profiles have been determined for most of these receptor complexes in heterologous expression systems (Christopoulos et al., 1999;Nagoshi et al., 2002;Hay et al., 2003Hay et al., , 2005Kuwasako et al., 2003a;Bailey and Hay, 2006 ABBREVIATIONS: AMY, amylin receptor phenotype; AM, adrenomedullin; BIBN4096BS, 1-piperidinecarboxamide, N-[2-[[5amino-l-[[4-(4- Hay et al., 2005). Such obs...

Spatial Epitope Prediction server for Protein Antigens (SEPPA) has received lots of feedback since being published in 2009. In this improved version, relative ASA preference of unit patch and consolidated amino acid index were added as further classification parameters in addition to unit-triangle propensity and clustering coefficient which were previously reported. Then logistic regression model was adopted instead of the previous simple additive one. Most importantly, subcellular localization of protein antigen and species of immune host were fully taken account to improve prediction. The result shows that AUC of 0.745 (5-fold cross-validation) is almost the baseline performance with no differentiation like all the other tools. Specifying subcellular localization of protein antigen and species of immune host will generally push the AUC up. Secretory protein immunized to mouse can push AUC to 0.823. In this version, the false positive rate has been largely decreased as well. As the first method which has considered the subcellular localization of protein antigen and species of immune host, SEPPA 2.0 shows obvious advantages over the other popular servers like SEPPA, PEPITO, DiscoTope-2, B-pred, Bpredictor and Epitopia in supporting more specific biological needs. SEPPA 2.0 can be accessed at http://badd.tongji.edu.cn/seppa/. Batch query is also supported.

Nonalcoholic steatohepatitis (NASH) is the most prevalent cause of chronic liver disease worldwide. Macrophagemediated inflammation plays a critical role in NASH pathogenesis; however, optimum therapies for macrophage activation and NASH remain elusive. HSPA12A encodes a novel member of the HSP70 family. Here, we report that NASH patients showed increased hepatic HSPA12A expression and serum HSPA12A contents. Intriguingly, knockout of HSPA12A (Hspa12a 2/2) in mice attenuated high-fat diet (HFD)-induced hepatic steatosis and injury. HFD-induced macrophage polarization toward an M1 phenotype and inflammatory responses in the liver of Hspa12a 2/2 mice were also attenuated. Loss-and gain-of-function studies revealed that the de novo lipogenesis in hepatocytes was regulated by the paracrine effects of macrophage HSPA12A rather than by hepatocyte HSPA12A. In-depth molecular analysis revealed that HSPA12A interacted with the M2 isoform of pyruvate kinase (PKM2) in macrophages and increased its nuclear translocation, thereby promoting M1 polarization and secretion of proinflammatory M1 cytokines; this led, ultimately, to hepatocyte steatosis via paracrine effects. Taken together, these findings show that HSPA12A acts as a novel regulator of M1 macrophage polarization and NASH pathogenesis by increasing nuclear PKM2. Strategies that inhibit macrophage HSPA12A might be a potential therapeutic intervention for NASH.

Alternative splicing expands proteome diversity to GPCRs. Distinct receptor variants have been identified for a secretin family GPCR, the calcitonin receptor (CTR). The possible functional contributions of these receptor variants are further altered by their potential interactions with receptor activity-modifying proteins (RAMPs). One variant of the human CTR lacks the first 47 residues at its N terminus [D(1-47)hCT(a)]. However, very little is known about the pharmacology of this variant or its ability to interact with RAMPs to form amylin receptors. EXPERIMENTAL APPROACHD(1-47)hCT(a) was characterized both with and without RAMPs in Cos7 and/or HEK293S cells. The receptor expression (ELISA assays) and function (cAMP and pERK1/2 assays) for up to six agonists and two antagonists were determined. KEY RESULTSDespite lacking 47 residues at the N terminus, D(1-47)hCT(a) was still able to express at the cell surface, but displayed a generalized reduction in peptide potency. D(1-47)hCT(a) retained its ability to interact with RAMP1 and formed a functional amylin receptor; this also appeared to be the case with RAMP3. On the other hand, its interaction with RAMP2 and resultant amylin receptor was reduced to a greater extent. CONCLUSIONS AND IMPLICATIONSD(1-47)hCT(a) acts as a functional receptor at the cell surface. It exhibits altered receptor function, depending on whether it associates with a RAMP and which RAMP it interacts with. Therefore, the presence of this variant in tissues will potentially contribute to altered peptide binding and signalling, depending on the RAMP distribution in tissues.

The traditional food supervision system has problems such as lack of industry chain and data fragmentation, which has caused the phenomenon of field regulation to be uncomprehensive in the existing regulatory system and the disposal of response lag. Combining the characteristics of blockchain such as distribution, transparent, and collegiality with the actual needs of regional autonomy, we propose the hierarchical multi-domain blockchain (HMDBC) network structure and the secondary-check mechanism, which can support timely correction and replacement of the malicious supervision nodes by regional nodes co-governance, auxiliary verification of supervision nodes, and arbitration of superior regions. In order to optimize the supervision nodes election, we proposed fuzzy comprehensive evaluation model of credibility, which can be used to objectively and fairly evaluate the comprehensive reputation of each node in that region by considering various influencing factors of node performance indicators. Furthermore, we designed the data block structure model, which can support the supervisor node replacement. Our system can perform automatic food quality detection and warning of substandard food in the entire industrial chain, with the use of smart contracts combined with the food industry standards. Finally, comparing the complexity of the traditional blockchain system with PBFT consortium, our system is more secure, with lower broadcast complexity.

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In order to investigate the relationship between gut microbiota and type 2 diabetic erectile dysfunction (T2DED), we analyzed the characteristics of gut microbiota in the Sprague-Dawley (SD) rats with T2DED. Thirty-five SD rats were randomly divided into two groups: control group (n=15) with normal diet, and experimental group (n=20) with construction of T2D model. Faecal and serum samples were collected at 2nd and 8th week after establishment of T2D model, respectively. Faecal samples were used for analysis of gut microbiota, and serum samples for detection of trimethylamine N-oxide (TMAO), lipopolysaccharide (LPS), and inflammatory factors like interleukin-1 (IL-1), IL-2, IL-10, and monocyte chemoattractantprotein-1 (MCP-1). The main compositions of gut microbiota were Bacteroidetes, Proteobacteria and Firmicutes at the phylum level, and Oscillospira, Allobaculum, Bacteroides, Ruminococcus, SMB53, Prevotella, Coprococcus, Sutterella and Blautia at the genus level with relatively higher abundance in all SD rats. The relative abundance of Enterococcus, Corynebacterium, Aerococcus, Facklamia (opportunistic pathogens in most case) increased, and that of Allobaculum, Bifidobacterium, Eubacterium, Anaerotruncus (beneficial bacteria) decreased in T2DED group as compared with that at 2nd week after establishment of T2D model (T2D2 group). The serum contents of TMAO, LPS, IL-1, IL-2, IL-10 and MCP-1 in T2DED group were significantly higher than those in control group. The gut microbiota of T2DED rats was inhibited. The gut microbiota of T2DED rats had changed, as the relative abundance of beneficial bacterium was decreased while that of opportunistic pathogens was increased. The variations of gut microbiota might lead to inflammation and prompt the emergence of erectile dysfunction in the rats with T2D. TMAO might play an important role in the formation of T2DED.