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Men with diabetic erectile dysfunction (ED) respond poorly to the currently available oral phosphodiesterase-5 inhibitors. Therefore, functional therapies for diabetic ED are needed. Stromal vascular fraction (SVF) and the adenovirus-mediated cartilage oligomeric matrix angiopoietin-1 (Ad-COMP-Ang1) gene are known to play critical roles in penile erection. We previously reported that SVF and Ad-COMP-Ang1 have only a short-term effect in restoring erectile function. Further improvements to ED therapy are needed for long-lasting effects. In the present study, we aimed to test if the combination of SVF and Ad-COMP-Ang1 could extend the erection effect in diabetic ED. We found that the combination therapy showed a long-term effect in restoring erectile function through enhanced penile endothelial and neural cell regeneration. Combination therapy with SVF and Ad-COMP-Ang1 notably restored cavernous endothelial cell numbers, pericyte numbers, endothelial cell-cell junctions, decreased cavernous endothelial cell permeability, and promoted neural regeneration for at least 4 weeks in diabetic mice. In summary, this is an initial description of the long-term effect of combination therapy with SVF and Ad-COMP-Ang1 in restoring erectile function through a dual effect on endothelial and neural cell regeneration. Such combination therapy may have therapeutic potential for the treatment of diabetic ED.

Background Acute gastrointestinal injury is associated with significantly increased mortality in critically ill patients. However, markers for measuring acute gastrointestinal injury are neither sensitive nor specific. Objective To determine whether enzymes in digestive fluid are predictive of the severity of acute gastrointestinal injury. Methods A prospective observational study was conducted between June 2015 and December 2015 in a surgical intensive care unit. Enrolled patients were classified by acute gastrointestinal injury grade according to the 2012 European Society of Intensive Care Medicine system. Digestive fluid was collected through nasointestinal tubes and analyzed 24 hours after the diagnosis of acute gastrointestinal injury. Intestinal markers of injury (pH, interleukin 6, interleukin 10, tumor necrosis factor α, and secretory immunoglobulin A) were measured in digestive fluid. Results Of the 76 patients included, acute gastrointestinal injury was grade I in 41, grade II in 20, grade III in 8, and grade IV in 7. Secretory immunoglobulin A was an independent predictor of grade III acute gastrointestinal injury. When data from patients with grades I and II injury and patients with grades III and IV injury were grouped together, analysis revealed that pH, interleukin 10, and secretory immunoglobulin A were independent predictors of acute gastrointestinal failure. Conclusions Secretory immunoglobulin A was predictive of grade III acute gastrointestinal injury. Digestive fluid markers of injury (pH, interleukin 10, and secretory immunoglobulin A) were predictors of acute gastrointestinal failure. Further study is required to determine if other markers are specific or sensitive for acute gastrointestinal injury.

Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti-inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide-induced cytotoxicity in murine leukemia WEHI-3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca2+ release and mitochondrial membrane potential (ΔΨm ), and activations of caspase-8, -9, and -3. Triptolide increased protein levels of Fas, Fas-L, Bax, cytochrome c, caspase-9, Endo G, Apaf-1, PARP, caspase-3 but reduced levels of AIF, ATF6α, ATF6β, and GRP78 in WEHI-3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC-3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross-talk between cross-interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac-3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide did not significant affect NK cell activities in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 550-568, 2017.

Overall scheme for large-aircraft Intelligence Virtual Maintenance Training System (IVMTS) information model which integrates the Standard for the Exchange of Product model data (STEP) standard geometric model with agent model is presented in this paper. Firstly, the system multi-agent model architecture are constructed. Secondly, Integrated STEP standard geometric model with agent model and maintenance applying information, the maintenance object agent IP(intelligence part) ,IB(intelligence body) and IT(intelligence tool) are constructed utilizing the EPRESS-G language. Thirdly, the communication method between components and system level agents is presented based on Blackboard model each other. Finally, how to realize the model under catia-caa secondary development method is described, and the intelligence maintenance process simulation, maintenance of the elements of the intelligent interaction, intelligence collision detection and maintenance sequence optimization simulation can be realized in this method in IVMTS.

This study reports the preparation of four varieties of water extract from sweet potato leaves from Taiwan, including TNG10, TNG57, TNG66 and YSP, and evaluates their antioxidative activity. The EC50 values (scavenging DPPH radicals) of TNG10, TNG57, TNG66 and YSP were 0.27 ± 0.01, 0.19 ± 0.01, 0.41 ± 0.02, and 0.31 ± 0.02 mg/ml, respectively, on a freeze-dry weight basis. The total phenolic contents of these water extracts were in the order: TNG57 > TNG10 > TNG66 > YSP. The TNG10 and TNG57 extracts exhibited better reducing power and scavenging effects of superoxide radicals than did TNG66 and YSP. At a concentration of 1 mg/ml, TNG10 and TNG57 significantly protected HaCaT cells from H2O2-induced cytotoxicity. The water extracts of YSP had more flavonoids than had those of TNG66 which may have contributed to their higher activity in many antioxidative assays. These results suggest that the water extracts of all four varieties of sweet potato leaves, and especially TNG10 and TNG57, display potent antioxidative effects.

This study showed that both water extracts (WAF-W) and ethanol extracts (EAF-W) of Auricularia fuscosuccinea (Montagne) Farlow, white strain (AF-W) demonstrated significantly stronger antioxidative effects than did commercially available Tremella fuciformis sporocarp extracts (WSK; with the exception of EAF-W in terms of superoxide radical scavenging activity levels). The moisture retention capacity of WAF-W is as potent as that of sodium hyaluronate (SHA), but less than that of WSK. No corrugation or fissures were observed in WAF-W film; only the SHA and WSK films demonstrated such effects in low-moisture conditions. The WAF-W solution also exhibited stable viscosity at high temperatures, indicating that the WAF-W film was more stable compared with the SHA and WSK films. WAF-W induced no adverse effects when a hen’s egg test was performed on the chorioallantoic membrane (CAM). This study demonstrated that WAF-W exhibits excellent potential as a topical material for skin moisturizing and anti-aging effects.

We investigated the kinetics of 4N-acetyl-pentapeptides, Ac-P1, Ac-P2, Ac-P3, and Ac-P4, regarding inhibition of mushroom tyrosinase activity. The peptides sequences of Ac-P1, Ac-P2, Ac-P3, and Ac-P4 were Ac-RSRFK, Ac-KSRFR, Ac-KSSFR, and Ac-RSRFS, respectively. The 4N-acetyl-pentapeptides were able to reduce the oxidation ofL-DOPA by tyrosinase in a dose-dependent manner. Of the 4N-acetyl-pentapeptides, only Ac-P4 exhibited lag time (80 s) at a concentration of 0.5 mg/mL. The tyrosinase inhibitory effects of Ac-P4 (IC500.29 mg/mL) were more effective than those of Ac-P1, Ac-P2, and Ac-P3, in which IC50s were 0.75 mg/mL, 0.78 mg/mL, and 0.81 mg/mL, respectively. Kinetic analysis demonstrated that all 4N-acetyl-pentapeptides were mixed-type tyrosinase inhibitors. Furthermore, 0.1 mg/mL of Ac-P4 exhibited significant melanogenesis inhibition on B16F10 melanoma cells and was more effective than kojic acid. The melanogenesis inhibition of Ac-P4 was dose-dependent and did not induce any cytotoxicity on B16F10 melanoma cells.

Oxidative stress, including Ultraviolet (UV) irradiation-induced skin damage, is involved in numerous diseases. This study demonstrates that water extract ofEclipta prostrataL. (WEP) has a potent effect in scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide radicals, and chelating ferrous ion, exhibiting IC50values of 0.23 mg/mL, 0.48 mg/mL, and 1.25 mg/mL, respectively. The WEP total phenol content was 176.45 mg gallic acid equivalents (GAE)/g sample. Chlorogenic acid, a component of the plant's active ingredients, was determined by HPLC and antioxidative assay. However, no caffeic acid, stigmasterol, or wedelolactone was present in WEP. WEP absorbs both UVA and UVB irradiation, and furthermore, the extract shows a dose-dependent response in the protection of HaCaT human keratinocytes and mouse fibroblasts 3T3 cells against UVB-induced cytotoxicity, which may result from a synergistic effect between chlorogenic acid and other active components present in WEP.

This is the first study to investigate the biological activities of fermented extracts ofRhodiola roseaL. (Crassulaceae) andLonicera japonicaThunb. (Caprifoliaceae).Alcaligenes piechaudiiCC-ESB2 fermented and ethanol extracts ofRhodiola roseaandLonicera japonicawere prepared and the antioxidative activities of different concentrations of samples were evaluated usingin vitroantioxidative assays. Tyrosinase inhibition was determined by using the dopachrome method with L-DOPA as substrate. The results demonstrated that inhibitory effects (ED50values) on mushroom tyrosinase of fermentedRhodiola rosea, fermentedLonicera japonica, ethanol extract ofLonicera japonica,and ethanol extract ofRhodiola roseawere 0.78, 4.07, 6.93, and >10 mg/ml, respectively. The DPPH scavenging effects of fermentedRhodiola rosea(ED50= 0.073 mg/ml) and fermentedLonicera japonica(ED50= 0.207 mg/ml) were stronger than effects of their respective ethanol extracts. Furthermore, the scavenging effect increases with the presence of high content of total phenol. However, the superoxide scavenging effects of fermentedRhodiola roseawas less than effects of fermentedLonicera japonica. The results indicated that fermentation ofRhodiola roseaandLonicera japonicacan be considered as an effective biochemical process for application in food, drug, and cosmetics.

We investigated the relationship between [Ca2+]i, myosin light chain (LC20) phosphorylation and isometric force in guinea pig aortic strips during contractions activated by a thromboxane A2 analogue, (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619). Isometric force and [Ca2+]i were measured simultaneously using preloaded aequorin as the intracellular calcium indicator. LC20 phosphorylation levels were determined by two dimensional polyacrylamide gel electrophoresis in parallel preparations. Contractions induced by U46619 were accompanied by increases in [Ca2+]i and LC20 phosphorylation. The chelation of extracellular calcium with 2.5 mM EGTA significantly inhibited U46619-induced increases in [Ca2+]i, isometric force and LC20 phosphorylation. Steady-state force assumed a similar dependence on LC20 phosphorylation for contractions stimulated by potassium depolarization, alpha 1-adrenergic agonist phenylephrine and U46619 either in the presence or absence of extracellular calcium. On the contrary, the [Ca2+]i/force relation revealed that both U46619 and phenylephrine stimulated greater isometric force at lower [Ca2+]i than did KCl depolarization. The addition of a protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), decreases force without significantly affecting either [Ca2+]i or LC20 phosphorylation levels. These results suggest that in guinea pig aortic smooth muscle U46619 increases the calcium sensitivity of the contractile apparatus but does not change the LC20 phosphorylation/force relation in comparison to K+ depolarization. Protein kinase C is activated during U46619-stimulated contraction and might be involved in mechanisms other than LC20 phosphorylation.

The native amino acid ergothioneine, a thiourea derivative of histidine, inhibits mushroom tyrosinase activity in a dose-dependent manner, with an IC(50) value of 1.025 mg/ml (4.47 mM). By contrast, histidine exhibited no inhibitory effect on mushroom tyrosinase activity. We characterized ergothioneine as a noncompetitive tyrosinase inhibitor using a Lineweaver-Burk plot of experimental kinetic data. The IC(50) value for ergothioneine scavenging of 2,2-diphenyl-1-picrylhydrazyl was 6.110 ± 0.305 mg/ml, much higher than the IC(50) for inhibition of tyrosinase activity which indicating ergothioneine on tyrosinase shows a weak correlation to its antioxidative activity. The results demonstrated that ergothioneine has a potent inhibition effect on tyrosinase enzyme activity, resulting from the presence of the sulfur substituted imidazole ring in ergothioneine.

AIM: To detect multiple H pylori antibodies in serum samples of individuals who carryH pylori by protein array. METHODS: Recombinant H pylori antigens, urease B subunit (UreB), vacuolating toxin A (VacA) and cytotoxin associated gene A protein (CagA), were prepared and immobilized in matrixes on nitrocellulose membrane by robotics to bind the specific immunoglobulin G (IgG) antibodies in serum. Staphylococcus protein A (SPA) labeled by colloid gold was used to integrate the immuno-complex and gave red color signal. The scanner based on charge-coupled device (CCD) could collect the image signal and convert it into digital signal. RESULTS: When human IgG was printed on the membrane in increasing concentrations and incubated with immunogold, a linear dose response curve was obtained and the detection limit for IgG was about 0.025 ng. The cutoff values, which were defined as the mean grey level plus 3 times of standard deviation, were 27.183, 28.546 and 27.402, for anti-UreB IgG, anti-CagA IgG and anti-VacA IgG, respectively, as 400 human serum samples with negative H pylori antibodies were detected by the protein array. When 180 serum samples from patients in hospital were employed for detection of IgG against UreB, CagA and VacA, the sensitivity of the protein array was 93.4%, 95.4%, 96.0%, and the specificity was 94.8%, 94.4% and 97.5%, respectively, as compared with the results obtained by ELISA. The assay also showed high reproducibility, uniformity and stability, and the results were available within 30 min. CONCLUSION: The protein array is a very practical method for rapid detection of multiple antibodies in serum samples. It is especially useful for large scale epidemiological investigation of the infection of H pylori.

AIM: H pylori genomes are highly diversified. This project was designed to genotype H pylori isolates by the polymerase chain reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) fingerprinting technique and to verify its stability by Southern blotting and DNA sequencing. METHODS: Clinical isolates of H pylori were cultured from gastric antra and cardia of 73 individuals, and genomic DNA was prepared for each isolate. RAPD was carried out under optimized conditions. 23S rDNA was regarded as an internal control, and a 361 bp rDNA fragment (RDF) was used as a probe to screen the RAPD products by Southern blotting. Ten RDFs from different clinical isolates and the flanking regions (both upstream and downstream) of four RDFs were amplified and sequenced. RESULTS: H pylori isolates from different individuals had different RAPD profiles, but the profiles for isolates cultured from different gastric sites of a given individual were identical in all but one case. Isolates from 27 individuals were RDF positive by Southern blotting. Sequences of the RDFs and their flanking regions were almost the same between the RDF positive and negative isolates as determined by Southern blotting. There was no binding site for random PCR primer inside the sequences. CONCLUSION: RAPD is very useful in genotyping H pylori grossly on a large scale. However, it seems unstable in amplification of low yield fragments, especially those that do not appear as visible bands on the agarose gel stained with EB, since the primer is partially matched to the template.

AIM: The genomes of Helicobacter pylori (H. pylori) from different individuals are different. This project was to identify the strain specific DNA sequences between two clinical H. pylori isolates by suppression subtractive hybridization (SSH). METHODS: Two clinical H. pylori isolates, one from gastric ulcer (GU, tester) and the other from non-ulcer dyspepsia (NUD, driver), were cultured and the genomic DNA was prepared and submitted to Alu I digestion. Then two different adaptors were ligated respectively to the 5’-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNA. The un-hybridized tester DNA sequences were amplified by two sequential PCR and cloned into pGEM-T-Easy Vector. The tester strain specific inserts were screened and disease related DNA sequences were identified by dot blotting. RESULTS: Among the 240 colonies randomly chosen, 50 contained the tester strain specific DNA sequences. Twenty three inserts were sequenced and the sizes ranged from 261 bp to 1036 bp. Fifteen inserts belonged to the H.pylori plasmid pHPO100 that is about 3.5 kb and codes a replication protein A. Other inserts had patches of homologous to the genes of H.pylori in GenBank. Various patterns of dot blots were given and no GU strain unique DNA sequences were found when 4 inserts were used as probes to screen the genomic DNA from 27 clinical isolates, 8 from GU, 12 from duodenum ulcer (DU), 4 from GU-DU, 2 from NUD and 1 from gastric cancer (GC). But a 670 bp DNA fragment (GU198) that was a bit homologous to the 3’-end of the gene of thymidylate kinase was positive in 7 GU strains (7/8), 3 GU-DU strains (3/4) and 3 DU strains (3/12). A 384 bp fragment (GU79) of the replication gene A (repA) was positive only in 4 H.pylori isolates, 2 from GU and 2 from GU-DU. CONCLUSION: Differences exist in the genes of different H.pylori isolates. SSH is very effective to screen H.pylori strain specific DNA sequences between two clinical isolates, and some of these sequences may have clinical significance.

Hepatitis C is a world-wide epidemic disease stemming from the hepatitis C virus (HCV). HCV is not only the pathogenic factor of hepatitis C but also plays an important role in the process of triggering and development of cirrhosis and liver cancer. There are two indicators for detecting HCV infection: one is HCV RNA which is the gene of the virus itself and the other is anti-HCV which is the antibody produced when the HCV antigen stimulates the body. The HCV antibody detection includes the detection of HCV IgG and HCV-IgM antibody. At present, EIA and the recombinant immunoblot assay (RIBA) are mainly used for detecting anti-HCV-IgG. By using the colloidal gold labeling technique to fix the structural and the non-structural domains of HCV recombinant antigen on the millipore membrane, a dot immunogold filtration assay for detecting anti-HCV-IgG was successfully established.

Helicobacter pylori (Hp) plays an important role in the upper digestive tract diseases. It can be divided into two main groups (toxic and non-toxic Hp) according to the production of vacuolating cytotoxin (VacA). The toxic bacteria also produce cytotoxin associated protein A (CagA) which might have something to do with the transcription, folding, transportation or the function of VacA. Studies showed that CagA positive Hp (CagA+Hp) accounted for more than 50% of all kinds of Hp, and peptic ulcer and gastric cancer were closely related to their infection[1-7]. Therefore, detection of the infection of CagA+Hp is of great significance. This study was carried out to express CagA gene in E.coli and develop an immuno-assay for the rapid detection of CagA+Hp.

The hepatitis A virus specific immunoglobulin M (IgM) antibody is a specific serological marker for early diagnosis of hepatitis A. At present, the methods used at home or abroad for detecting anti-HAV IgM are RIA, ELISA and SPHAI. The dot immunogold combination assay that has been developed since 1989 is a new technique with the property of simple and rapid immunological detection, by using the red colloidal gold particles to label the antibodies as indicator, and the millipore filtering membrane coated with antigen as the carrier. Affected by filtration and condensation, the antigen antibody reaction is enabled to go on rapidly. When the reaction is positive, red dots appear on the membrane. It takes about 2 min to 4 min for the whole reaction to be carried out. With the above technique, we have established the dot immunogold filtration assay (DIGFA) for rapid detection of anti-HAV IgM with comparatively satisfactory results.