Quantitative proteomics reveals key pathways in the symbiotic interface and the likely extracellular property of soybean symbiosome.

An effective symbiosis between legumes and rhizobia relies largely on diverse proteins at the plant-rhizobium interface for material transportation and signal transduction during symbiotic nitrogen fixation. Here, we report a comprehensive proteome atlas of the soybean symbiosome membrane (SM), peribacteroid space (PBS), and root microsomal fraction (RMF) using state-of-the-art label-free quantitative proteomic technology. In total, 1759 soybean proteins with diverse functions are detected in the SM, and 1476 soybean proteins and 369 rhizobial proteins are detected in the PBS. The diversity of SM proteins detected suggests multiple origins of the SM. Quantitative comparative analysis highlights amino acid metabolism and nutrient uptake in the SM, indicative of the key pathways in nitrogen assimilation. The detection of soybean secretory proteins in the PBS and receptor-like kinases in the SM provides evidence for the likely extracellular property of the symbiosome and the potential signaling communication between both symbionts at the symbiotic interface. Our proteomic data provide clues for how some of the sophisticated regulation between soybean and rhizobium at the symbiotic interface is achieved, and suggest approaches for symbiosis engineering.

POD1-SUN-CRT3 chaperone complex guards the ER sorting of LRR receptor kinases in Arabidopsis.

Protein sorting in the secretory pathway is essential for cellular compartmentalization and homeostasis in eukaryotic cells. The endoplasmic reticulum (ER) is the biosynthetic and folding factory of secretory cargo proteins. The cargo transport from the ER to the Golgi is highly selective, but the molecular mechanism for the sorting specificity is unclear. Here, we report that three ER membrane localized proteins, SUN3, SUN4 and SUN5, regulate ER sorting of leucine-rich repeat receptor kinases (LRR-RKs) to the plasma membrane. The triple mutant sun3/4/5 displays mis-sorting of these cargo proteins to acidic compartments and therefore impairs the growth of pollen tubes and the whole plant. Furthermore, the extracellular LRR domain of LRR-RKs is responsible for the correct sorting. Together, this study reports a mechanism that is important for the sorting of cell surface receptors.

First-principles calculations of high-pressure physical properties anisotropy for magnesite.

The first-principles calculations based on density functional theory with projector-augmented wave are used to study the anisotropy of elastic modulus, mechanical hardness, minimum thermal conductivity, acoustic velocity and thermal expansion of magnesite (MgCO3) under deep mantle pressure. The calculation results of the phase transition pressure, equation of state, elastic constants, elastic moduli, elastic wave velocities and thermal expansion coefficient are consistent with those determined experimentally. The research results show that the elastic moduli have strong anisotropy, the mechanical hardness gradually softens with increasing pressure, the conduction velocity of heat in the [100] direction is faster than that in the [001] direction, the plane wave velocity anisotropy first increases and then gradually decreases with increasing pressure, and the shear wave velocity anisotropy increases with the increase of pressure, the thermal expansion in the [100] direction is greater than that in the [001] direction. The research results are of great significance to people's understanding of the high-pressure physical properties of carbonates in the deep mantle.

Hepatoid adenocarcinoma of the stomach: Thirteen case reports and review of literature.

BACKGROUND: The aim of the present study was to examine the clinical characteristics of hepatoid adenocarcinoma of the stomach (HAS) and its diagnosis, treatment, and prognosis. CASE SUMMARY: A retrospective analysis of 13 HAS cases was performed. The mean age of the 13 patients was 66.08 years, and 10 of the 13 patients were male. Prior to treatment, the alpha-fetoprotein levels in the serum were elevated in 7 patients, the tumour was located in the distal or gastric body in 11 patients, and the gastroscopy pathological results showed that 3 patients had poorly differentiated tumours and that 8 patients had moderately/poorly differentiated tumours. Abdominal CT scans showed local stomach wall thickening, and enlarged lymph nodes were visible around the stomach in 8 patients. Of the 13 patients, 11 underwent radical surgery. The clinical pathological staging was as follows: Stage II in 2 cases; stage III in 8 cases; and stage IV in 1 case. A total of 3 patients were lost to follow-up. Otherwise, as of the last follow-up, 3 patients had survived for 56 mo, and the other 7 patients failed to achieve long-term survival (survival period of 1-56 mo). CONCLUSION: HAS is a special type of gastric cancer, and the prognosis of HAS has improved compared with past prognoses. Measurement of alpha-fetoprotein, early diagnosis, active surgical treatment, and application of new diagnostic and treatment techniques are conducive to improving the prognosis of HAS.

Curculigoside facilitates fear extinction and prevents depression-like behaviors in a mouse learned helplessness model through increasing hippocampal BDNF.

Curculigoside (CUR) is the main active component of traditional Chinese medicine Curculigoorchioides Gaertn (Xianmao in Chinese), which exhibits a variety of pharmacological activities. In this study we investigated the effects of CUR on fear extinction and related depression-like behaviors in mice. In fear conditioning task, we found that administration of CUR (1.6, 8, 40 mg·kg-1·d-1, ip, for 7 days) did not affect memory consolidation, but CUR at higher doses (8, 40 mg·kg-1·d-1) significantly facilitated fear extinction, especially on D3 and D4. Moreover, CUR administration significantly ameliorated the fear conditioning-induced depression-like behaviors, likely through promoting fear extinction. We showed that CUR increased the expression of brain-derived neurotrophic factor (BDNF) and phosphorylation of tropomyosin receptor kinase B (TrkB) in the hippocampus, and activated protein kinase B (Akt)-mammalian target of the rapamycin (mTOR) signaling pathway. Administration of the selective TrkB agonist 7,8-dihydroxyflavone (7,8-DHF, 5 mg·kg-1·d-1, ip) also facilitated fear extinction, ameliorated depression-like behaviors. We established a mouse learned helplessness (LH) model to evaluate the antidepressant activity of CUR. The spatial memory was assessed in Morris water maze. We showed that LH-induced depression-like behaviors, including prolonged immobility times in forced swim and tail suspension tests as well as spatial memory impairments; LH also downregulated BDNF expression and the Akt-mTOR signaling pathway in the hippocampus. Administration of CUR (1.6, 8, 40 mg·kg-1·d-1, ip, for 14 days) or 7,8-DHF (5 mg·kg-1·d-1, ip, for 3 days) prevented LH-induced depression-like behaviors and promoted BDNF expression and the Akt-mTOR signaling pathway. In conclusion, CUR can accelerate the fear memory extinction and ameliorate depression-like behaviors in mice via promoting BDNF expression and activating the Akt-mTOR signaling pathway in the hippocampus.

Novel anti-inflammatory target of geniposide: Inhibiting Itgß1/Ras-Erk1/2 signal pathway via the miRNA-124a in rheumatoid arthritis synovial fibroblasts.

Geniposide (GE) is an active component isolated from the fruit of Gardenia jasminoides Ellis that has anti-inflammatory and other pharmacological effects; however, the underlying mechanism of GE action has not been elucidated in rheumatoid arthritis (RA). Previous studies have shown that GE plays a therapeutic role in RA via regulation of the integrin beta 1 (Itgß1)-mediated Ras-Erk1/2 signalling pathway. However, the specific mechanism of GE action on Itgß1 has not been clarified. Recent evidence indicates that microRNAs (miRNAs) are involved in the development of RA. In this study, we developed a miRNA-124a-based synoviocyte repair strategy. We demonstrated that miRNA-124a can directly inhibit the expression of the Itgß1 gene and decrease TNF-α-stimulated cell proliferation in vitro. MH7A cells were obtained from the patient with RA and treated with GE in the presence of TNF-α (10 ng/mL). Additionally, we demonstrated that the expression of miRNA-124a can be regulated by GE. GE upregulated the expression of miRNA-124a and decreased the expression of Itgß1 at the mRNA and protein levels. The results of the present study are the first to suggest that GE inhibits TNF-α-stimulated cell proliferation and blocks the activation of the Ras-Erk1/2 pathway via the upregulation of miRNA-124a expression. Our study elucidates the role of miRNA-124a as a protected miRNA in RA and may provide a novel strategy for the diagnosis and treatment of RA in the future.

Quantitative Analysis of Multi-components by Single Marker and Fingerprint Analysis of Achyranthes bidentata Blume.

A simple and effective method of high performance liquid chromatography (HPLC) with diode array detection was established to identify the origin of Achyranthes bidentata Blume and evaluate its quality, based on chromatographic fingerprint combined with the similarity analysis, hierarchical cluster analysis and the quantitative analysis of multi-components by single marker (QAMS). In the chromatographic fingerprint, 16 peaks were selected as the common model to evaluate the similarities among 18 batches (S1-S18) of A. bidentata Blume samples collected from different origins in China. The similarities values for 18 batches of samples were more than 0.75, which compared with control fingerprint. Furthermore, 18 batches of A. bidentata Blume samples were categorized into two groups for quantitative analysis, the quantification of three bioactive constituents (ß-ecdysterone, cyasterone and 5-hydroxymethyl furfural) between QAMS and external standard method proved the consistency of the two methods, the three constituents showed good regression (R > 0.9995) within linear ranges, and their recoveries were within the range of 97.6-101.5%. This study demonstrated that the quality of A. bidentata Blume can be successfully evaluated by means of a combination of HPLC chromatographic fingerprint and QAMS approach.

Anti-inflammatory Mechanism of Geniposide: Inhibiting the Hyperpermeability of Fibroblast-Like Synoviocytes via the RhoA/p38MAPK/NF-κB/F-Actin Signal Pathway.

Geniposide (GE) is the extraction and purification of iridoid glycosides from the Gardenia jasminoides Ellis, which is a promising anti-inflammatory drug, but its mechanism of actions on rheumatoid arthritis (RA) has not been clarified. This study investigated the molecular mechanism behind GE reduced the high permeability of fibroblast-like synoviocytes (FLSs) derived from SD rats with adjuvant arthritis (AA), with the aims of observing the action of GE in AA rats and exploring new therapeutic strategies for RA treatment. The CCK-8 method was used to detect FLSs proliferation. The pro-inflammatory cytokines levels and anti-inflammatory cytokines levels in FLSs were determined by ELISA kits. FLSs permeability assay was performed on Transwell. Immunofluorescence was used to assay the arrangement and morphology of F-actin. The expression of the key molecules related to FLSs permeability (RhoA, p-p38MAPK, NF-κB p-p65 and F-actin) was detected by western blotting. After treatment with lipopolysaccharide (LPS), the proliferation and the permeability of the cells increased significantly (all P < 0.05). The expression of RhoA, p-p38MAPK, NF-κB p-p65 and F-actin in FLSs was higher compared with the control group, and F-actin was redistributed, with the formation of additional stress fibers. But, these conditions were moderated after treatment with GE. We demonstrated that the treatment of different concentrations of GE (25, 50, and 100 µg/mL) had a significant inhibitory effect on the proliferation and permeability of FLSs in vitro. Furthermore, the levels of interleukin (IL)-1ß and IL-17 secreted by FLSs were decreased in different doses of GE groups, and the levels of anti-inflammatory cytokines (IL-4, TGF-ß1) were increased. Under treatment with GE, low expression of RhoA downregulated expression of p-p38MAPK, NF-κB p-p65, and F-actin while compared with control group, and restored the hyperpermeability of FLSs due to LPS treatment. Taken together, GE might play its anti-inflammatory and immunoregulatory effects via regulating the relative equilibrium of pro-inflammatory cytokines and anti-inflammatory cytokines. GE attenuated the hyperpermeability of FLSs. The down-regulation of the conduction of RhoA/p38MAPK/NF-κB/F-actin signal may play a critical role in the mechanisms of GE on RA. GE could be an effective therapeutic agent for the treatment of RA.

Microdialysis sampling combined with ultra-high-performance liquid chromatography/tandem mass spectrometry for the determination of geniposide in dialysate of joint cavities in adjuvant arthritis rats.

RATIONALE: Microdialysis has been used to detect the concentrations of drugs in tissues. Geniposide (GE), an iridoid glycoside compound, is the main bioactive component of Gardenia jasminoides Ellis fruit. We previously demonstrated that GE could control the activity of cytokines and reduce levels of inflammation in adjuvant arthritis (AA) rats, but the topic of concentration changes over time in the joint synovia of AA has rarely been studied. METHODS: In this study, a microdialysis technique combined with ultra-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UHPLC/ESI-MS/MS) was set up and confirmed to assay GE in the dialysate of the joint cavity in AA rats. Mass detection was conducted in multiple reaction monitoring (MRM) mode with negative ESI, and paeoniflorin (Pae) was used as an internal standard (IS). RESULTS: A lower limit of quantitation (LLOQ) of 5 ng/mL was found using this method and with good linearity in the range of 5-4000 ng/mL. All the validation data including accuracy, precision, intra and inter-day repeatability and stability meet the requirements. The relative recoveries of GE were determined at approximately 40.01%. CONCLUSIONS: The measurements based on microdialysis combined with UHPLC/ESI-MS/MS provide a method for sampling and rapid sensitive analysis of GE in the dialysate of the joint cavity in AA rats. This method should be considered for future pharmacokinetics studies.

Immune Tolerance Effect in Mesenteric Lymph Node Lymphocytes of Geniposide on Adjuvant Arthritis Rats.

Rheumatoid arthritis (RA) is a systemic, Th1 cytokine-predominant autoimmune disease result in a chronic and inflammatory disorder. Geniposide (GE), an iridoid glycoside compound that is purified from Gardenia jasminoides Ellis, has antiinflammatory and other immunoregulatory effects, but its exact mechanism of actions on RA is unknown. The aim of this study was to elucidate antiinflammation effects of GE on adjuvant arthritis (AA) rats and its possible immune tolerance mechanisms. Male Sprague-Dawley rats were administered with GE (30, 60, and 120 mg/kg) orally from day 17 to 24 after immunization. Lymphocyte proliferation was assessed by MTT. Levels of interleukin-2 (IL-2), IL-4, and transforming growth factor-ß1 were tested by ELISA. The expression of ß2-AR, GRK2, and ß-arrestin-1 and ß-arrestin-2 was detected by western blot. Geniposide was found to relieve the secondary hind paw swelling and arthritis scores, along with attenuating histopathologic changes and decreasing IL-2 and increasing IL-4, transforming growth factor-ß1 in mesenteric lymph node (MLN) lymphocytes of AA rats. In addition, GE in vivo increased the expression of ß2-AR and decreased the expression of GRK2, ß-arrestin-1 and ß-arrestin-2, and level of cyclic adenosine monophosphate of MLN lymphocytes in AA rats. From these results, we can infer that GE on immune tolerance effects, ß2-AR desensitization, and ß2-AR-AC-cyclic adenosine monophosphate transmembrane signal transduction of MLN lymphocytes plays crucial roles in antiinflammatory and immunoregulatory pathogeneses of RA. Copyright © 2017 John Wiley & Sons, Ltd.

Antiinflammation Effects and Mechanisms Study of Geniposide on Rats with Collagen-Induced Arthritis.

Geniposide (GE), an iridoid glycoside compound purified from Gardenia jasminoides Ellis, has antiinflammatory and other pharmacological effects, but its mechanism of actions on rheumatoid arthritis (RA) have not been clarified. The purpose of this article was to investigate the pharmacological effects of GE on collagen-induced arthritis (CIA) rats and its feasible mechanisms. Collagen-induced arthritis was induced by injection of chicken type II collagen emulsion. The rats were orally administered with GE (33, 66, and 132 mg/kg) from days 14 to 30 after immunization. The histological examination showed that GE could attenuate histopathologic changes of mesenteric lymph node (MLN) in CIA rats. Geniposide inhibited the production of Interleukin 6 (IL-6) and IL-17, while promoting the production of IL-4 and transforming growth factor-beta 1 in MLN lymphocytes (MLNLs). Moreover, the proliferation capability of MLNLs was increased after the administration of GE. In addition, the treatment with GE in vivo decreased the expressions of P-Raf, P-MEK, and P-Erk1/2 in MLNLs. These results may highlight the antiinflammatory effects and possible mechanisms of GE in MLNLs of RA. Copyright © 2017 John Wiley & Sons, Ltd.

Genome-Wide Identification and Expression Analysis of the Tubby-Like Protein Family in the Malus domestica Genome.

Tubby-like proteins (TLPs), which have a highly conserved ß barrel tubby domain, have been found to be associated with some animal-specific characteristics. In the plant kingdom, more than 10 TLP family members were identified in Arabidopsis, rice and maize, and they were found to be involved in responses to stress. The publication of the apple genome makes it feasible to systematically study the TLP family in apple. In this investigation, nine TLP encoding genes (TLPs for short) were identified. When combined with the TLPs from other plant species, the TLPs were divided into three groups (group A, B, and C). Most plant TLP members in group A contained an additional F-box domain at the N-terminus. However, no common domain was identified other than tubby domain either in group B or in group C. An analysis of the tubby domains of MdTLPs identified three types of conserved motifs. Motif 1 and 2, the signature motifs in the confirmed TLPs, were always present in MdTLPs, while motif 3 was absent from group B. Homology modeling indicated that the tubby domain of most MdTLPs had a closed ß barrel, as in animal tubby domains. Expression profiling revealed that the MdTLP genes were expressed in multiple organs and were abundant in roots, stems, and leaves but low in flowers. An analysis of cis-acting elements showed that elements related to the stress response were prevalent in the promoter sequences of MdTLPs. Expression profiling by qRT-PCR indicated that almost all MdTLPs were up-regulated at some extent under abiotic stress, exogenous ABA and H2O2 treatments in leaves and roots, though different MdTLP members exhibited differently in leaves and roots. The results and information above may provide a basis for further investigation of TLP function in plants.

Design, synthesis and preliminary evaluation of the anti-inflammatory of the specific selective targeting druggable enzymome cyclooxygenase-2 (COX-2) small molecule.

CONTEXT: Development of a reliable and selective anti-inflammatory agent of cyclooxygenase-2 (COX-2), induced or up-regulated by inflammatory/injury stimulus such as IL-1ß, TNF-α and LPS in the various types of organs, tissues and cells, with low side effects is a long-standing medicinal chemistry problem with significant social implications. OBJECTIVE: To target druggable enzymome COX-2 by exploiting NSAIDs and genipin (GEP) in anti-inflammatory infection. MATERIALS AND METHODS: The compound aspirin GEP ester (AGE) was designed by computer-assisted screening, synthesized in the esterification of the acylate derivative and the methylate derivative with Et3N, and evaluated with 20, 40 and 60 mg/kg from days 18 to 24 after immunization in collagen-induced arthritis (CIA) rats by the sequential enzymatic experiments, western-blot analysis and pathological observation methods. RESULTS: AGE exhibited higher binding affinity with COX-1 and displayed the lowest estimated free energy with COX-2 than other ligands built by hanging NSAIDs with GEP, and was characterized by 1H NMR, 13C NMR and HRMS. AGE was competed against COX-2 with molecule-dependent potencies and selectivity (IC50: 0.36 mM; selectivity index: 275) in the sequential enzymatic experiments and decreased the expression of COX-2 in peripheral blood lymphocytes of CIA rats. AGE (40 and 60 mg/kg) could significantly relieve the secondary hind paw swelling and arthritis index, along with observing AGE attenuated histopathological changes of fibroblast like synovial tissue (FLST) and mesenteric lymph node lymphocytes (MLNL) in CIA rats. DISCUSSION AND CONCLUSION: AGE pharmacophore reported herein may be an effective strategy to develop a novel anti-inflammatory agent and potential inhibitor of COX-2.

Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome.

The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants.

[Plaster combined with splint for the treatment of gartland type III humeral supracondylar fractures in children].

OBJECTIVE: To study clinical effects of plaster combined with splint for the treatment of Gartland type 1I humeral fractures. METHODS: From March 2002 to May 2006, 24 children with humeral supracondylar fractures of Gartland type ill were reviewed. Among the patients, 14 patients were male and 10 patients were female, ranging in age from 4 to 12 years, averaged 6.6 years. Ten patients had injuries in the left and other 14 patients had injuries in the right limb. Firstly, the patients were treated with manipulative reduction to maintain the length of humerus without emphasis on anatomic reduction. Then, the patients were treated with external fixation using plaster for 5 to 7 days, and secondary manipulative reduction after swelling disappeared. Lastly, the patients were treated with external fixation using splint for 4 to 5 weeks until fracture union. RESULTS: All the patients were followed up, and the duration ranged from 5 months to 2 years, with an average of 1.2 years. All the patients had no complications such as neurovascular injury, myositis ossificans, forearm compartment syndrome and Volkmann contracture. According to ZHU Xiao-ting evaluation criteria for humeral supracondylar fractures in children, 12 patients got an excellent result, 8 good, 3 poor and 1 bad. CONCLUSION: Treatment of child humeral supracondylar fractures with plaster and splint has several advantages such as avoiding serious soft tissue injuries around fractures due to many times reduction, fracture dynamic correction to obtain satisfactory reduction, reducing complications, obtaining good reduction, and restoring elbow function in a relatively short period of time.