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Background N-methyl-d-aspartate receptors (NMDARs) are considered to be involved in several physiological and pathophysiological processes in addition to the progression of neurological disorders. However, how NMDARs are involved in the glycolytic phenotype of M1 macrophage polarization and the possibility of using them as a bio-imaging probe for macrophage-mediated inflammation remain unclear. Methods We analyzed cellular responses to NMDAR antagonism and small interfering RNAs using mouse bone marrow-derived macrophages (BMDMs) treated with lipopolysaccharide (LPS). An NMDAR targeting imaging probe, N-TIP, was produced via the introduction of NMDAR antibody and the infrared fluorescent dye FSD Fluor™ 647. N-TIP binding efficiency was tested in intact and LPS-stimulated BMDMs. N-TIP was intravenously administered to mice with carrageenan (CG)- and LPS-induced paw edema, and in vivo fluorescence imaging was conducted. The anti-inflammatory effects of dexamethasone were evaluated using the N-TIP-mediated macrophage imaging technique. Results NMDARs were overexpressed in LPS-treated macrophages, subsequently inducing M1 macrophage polarization. Mechanistically, NMDAR-mediated Ca2+ accumulation resulted in LPS-stimulated glycolysis via upregulation of PI3K/AKT/mTORC1 signaling. In vivo fluorescence imaging with N-TIP showed LPS- and CG-induced inflamed lesions at 5 h post-inflammation, and the inflamed lesions could be detected until 24 h. Furthermore, our N-TIP-mediated macrophage imaging technique helped successfully visualize the anti-inflammatory effects of dexamethasone in mice with inflammation. Conclusion This study demonstrates that NMDAR-mediated glycolysis plays a critical role in M1 macrophage-related inflammation. Moreover, our results suggest that NMDAR targeting imaging probe may be useful in research on inflammatory response in vivo.

Group 2 innate lymphoid cells (ILC2s) contribute to the pathogenesis of eosinophilic chronic rhinosinusitis with nasal polyps (CRSwNPs). However, the role of other subsets of ILCs and the differentiation of ILCs in CRSwNPs is not well understood. This study aimed to characterize the ILC subsets and evaluate the differentiation of ILCs from ILC precursors (ILCPs) in NP tissue.ILC subsets and ILCPs were evaluated by flow cytometry in fresh sinonasal mucosa from patients with CRSwNPs and control subjects. Subsets were compared based on clinical variables and immunological features of the patients. Sorted ILCPs (LinThe frequency of ILC1s and IFN-γ-producing ILC1s increased in non-eosinophilic NPs, whereas that of ILC2s and IL-5-producing ILC2s increased in eosinophilic NPs, particularly in patients with comorbid asthma. The frequency of ILC1s and IFN-γ-producing ILC1s, and frequency of ILC2s and IL-5-producing ILC2s positively correlated with that of neutrophils and eosinophils, respectively. The proportion of IFN-γ-producing ILC1s positively correlated with clinical severity and levels of IFN-γ and IL-8. The proportion of IL-5-producing ILC2s positively correlated with levels of IL-5, CCL24, and total IgE. ILCPs were identified in NP tissue and differentiated into IFN-γ-producing or IL-5-producing ILCs in response to increased IL-12 and IL-18 or IL-25 and IL-33 in non-eosinophilic NPs and eosinophilic NPs, respectively.ILC1s and ILC2s may be associated with neutrophilic and eosinophilic inflammation in CRSwNPs, respectively. In addition, ILCPs located in the sinus mucosa could differentiate into IFN-γ- or IL-5-producing cells in response to local cytokine stimuli.

Thioacetamide (TAA) was administered orally at 0, 10, and 30 mg/kg bw daily to Sprague-Dawley rats aged 6-7 weeks for 28 consecutive days. The bodyweight decreased, however, the relative organ weights including the liver and kidneys increased. No significant histopathologic abnormalities were found in the kidneys. The numbers of monocytes and platelets were significantly increased, however, the mean corpuscular volume and hematocrit values were decreased significantly in rats exposed to 30 mg/kg bw TAA. The expression levels of Kim-1 and NGAL were increased 4 to 5-fold in the kidney tissue of rats exposed to TAA, resulting in significant nephrotoxicity. Proteomic analysis was conducted and a total of 5221 proteins spots were resolved. Of these, three and 21 protein spots were up- and downregulated in a dose-dependent manner, respectively. The validation of seven proteins was performed by Western blot analysis. The expression level of ASAP2 was significantly upregulated, whereas RGS14, MAP7Dl, IL-3Rα, Tmod1, NQO2, and MUP were reduced. Sixteen isoforms of MUP were found by the 2DE immunoblot assay and were significantly downregulated with increasing exposure to TAA. MUP isoforms were compared in the liver, kidneys, and urine of untreated rats and a total of 43 isoforms were found.

One subtype of chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by the development of a T-helper type 2 (Th2) response and eosinophilic infiltration. Here, we aimed to establish an eosinophilic CRSwNP murine model, which would be essential to understand the underlying pathogenesis and establish a treatment strategy. C57BL/6 mice were challenged intranasally with a mixture of an Aspergillus oryzae-derived protease (AP) and ovalbumin (OVA) for 6, 8, or 12 consecutive weeks (12 mice/group); control mice received the same volume of phosphate-buffered saline for 12 weeks (n = 12). Sinonasal samples were evaluated histologically, and interleukin (IL)-4, IL-5, IL-13, eotaxin, keratinocyte chemoattractant, and macrophage inflammatory protein-2 mRNA levels in sinonasal mucosa were measured by real-time PCR. Protein levels of Th2 cytokines, INF-γ, IL-17A, and chemokines in nasal lavage fluid, and total serum IgE were measured by ELISA. Greater eosinophil infiltration in the subepithelial layer was observed in the challenged groups, compared with the control group. Polypoid mucosal lesions were predominantly observed in the 12-week group, which also exhibited mucosal thickening on micro-CT scans. The IL-4, IL-5, and IL-13 mRNA and protein levels were elevated in the sinonasal mucosa and nasal lavage fluid. INF-γ and IL-17A were undetectable or not elevated relative to the control group levels. In contrast, eotaxin levels were particularly elevated in the sinonasal mucosa and nasal lavage fluid in the 12-week group. In conclusion, intranasal AP and OVA exposure successfully induced Th2-specific CRSwNP in a murine model.

Life-Cycle Assessment has been used extensively in the construction industry to assess the environmental impacts of building materials. Attributional LCA considers processes in a supply chain which allows users to identify a process to improve to minimize the environmental impacts. However, the level of detail adopted in traditional attributional LCA is aggregate, not appropriate for process improvement efforts in the construction project context which is characterized as a complex system. This paper proposes Activity-based LCA (ABLCA) which adopts the methodology of the activity-based costing system to carry out the assessment and analysis of environmental impacts for the life cycle. The research carried out a case study on the curtain wall supply chain. The outcome of inventory analysis for each activity and environmental impact assessment showed the curtain wall supply chain process made an impact on five environmental impact categories: global warming air, acidification air, HH criteria air; eutrophication air, and photochemical smog air. With comparison to the outcome of environmental impact assessment from existing LCA, the proposed management system to investigate environmental impacts was addressed. The proposed ABLCA enables management to develop an environmental-impacts-reduction plan focusing on critical activities.

The present study investigated benexate hydrochloride betadex (BHB)-mediated ulcer healing, and changes to microcirculation modulated through nitric oxide synthase (NOS) and anti-inflammatory activity. A rat model of gastric mucosal injury was established through injection of a 60% acetic acid solution into the stomach. Following ulcer induction, the rats were administered BHB orally for 5 days at doses of 0, 100, 300 or 1,000 mg/kg. The highest dose of BHB was also administered with or without L-NG-nitroarginine methyl ester (L-NAME). The area of gastric ulcers was determined by planimetry, and expression of cyclooxygenases (COX), cytokines and NOS in stomach tissues were measured using western blotting. Compared with the control group, gastric ulcer size was significantly decreased in the 1,000 mg/kg BHB-treated group (P

Type I interferon (IFN-I, including IFN-α and IFN-β) response has been implicated in eosinophilic inflammation, in addition to antiviral function. This study aimed to investigate the role of IFN-I in the pathogenesis of eosinophilic chronic rhinosinusitis (ECRS). IFN-α, IFN-β, cytokine expression, and IFN-β cellular localization in the sinonasal tissue from control subjects and ECRS patients with nasal polyps (NP) were determined using real time-PCR, ELISA, and immunohistochemistry. ECRS was induced in wild-type (WT) and IFNAR1 knockout (Ifnar1 -/-) mice by intranasal challenge with Aspergillus protease and ovalbumin. Stromal cells cultured from NP tissue were stimulated by exogenous IFN-β, and their CCL11 production and IRF3, IRF7, STAT1, STAT2, and IRF9 gene and/or protein expression were measured. IFN-β, IL-5, IL-13, and CCL11 expression was higher in the NP tissue from ECRS patients, compared to the control group. IFN-β was highly colocalized with the CD11c+ cells in NP. IFN-β levels positively correlated with IL-5, IL-13, and CCL11 levels as well as the number of eosinophils in the NP tissue and CT score. The histological severity of ECRS, levels of IL-4, IL-5, IL-13, and CCL11 in the nasal lavage fluid, and total serum IgE levels were less in Ifnar1 -/- mice than in WT mice. CCL11 production, and STAT1 and STAT2 mRNA and STAT1, phospho-STAT1, and phospho-STAT2 protein expression were significantly increased by exogenous IFN-β in NP stromal cells. Our data suggest that IFN-β response was upregulated in ECRS and may play role in ECRS development. IFN-β may contribute to ECRS by enhancing CCL11 production. Thus, increased IFN-β response in the sinonasal mucosa may underlie ECRS pathogenesis.

Biofilm formation increases both the survival and infectivity of Vibrio cholerae, the causative agent of cholera. V. cholerae is capable of forming biofilms on solid surfaces and at the air-liquid interface, termed pellicles. Known components of the extracellular matrix include the matrix proteins Bap1, RbmA, and RbmC, an exopolysaccharide termed Vibrio polysaccharide, and DNA. In this work, we examined a rugose strain of V. cholerae and its mutants unable to produce matrix proteins by interfacial rheology to compare the evolution of pellicle elasticity in real time to understand the molecular basis of matrix protein contributions to pellicle integrity and elasticity. Together with electron micrographs, visual inspection, and contact angle measurements of the pellicles, we defined distinct contributions of the matrix proteins to pellicle morphology, microscale architecture, and mechanical properties. Furthermore, we discovered that Bap1 is uniquely required for the maintenance of the mechanical strength of the pellicle over time and contributes to the hydrophobicity of the pellicle. Thus, Bap1 presents an important matrix component to target in the prevention and dispersal of V. cholerae biofilms.

Aims Polaprezinc (PZ), which consists of l -carnosine and zinc, is widely used to treat gastric ulcers. We compared the effects of PZ with those of rebamipide (RM) on the expression of inflammatory cytokines, antioxidants, growth factors, and heat shock proteins (HSP) in a rat model. Main methods Seventy Sprague–Dawley rats were randomly assigned to test groups according to the dose of PZ at 5, 10, or 30 mg/kg or RM at 10, 30, or 100 mg/kg. Next, we obtained ulcer indices from rats with ethanol-induced gastric mucosal damage. Western blot analysis was used to evaluate the expression of various target proteins. Key findings Pathological ulcer indices in the PZ and RM groups were significantly lower than those in the control group. The levels of inflammatory cytokines (interleukin 1β [IL-1β], IL-6, IL-8, and tumor necrosis factor α) decreased, whereas the levels of platelet-derived growth factor-B, vascular endothelial growth factor, and nerve growth factor significantly increased after PZ administration. Furthermore, the expression of antioxidants (superoxide dismutase 1 [SOD-1], SOD-2, heme oxygenase-1, glutathione S-transferase, peroxidredoxin-1, and peroxidredoxin-5) was significantly higher in the PZ group, and the levels of HSP 90, 70, 60, 47, 27, and 10 significantly increased with an increase in PZ dose. Significance In a rat model of ethanol-induced gastric mucosal damage, PZ administration ameliorated ethanol-induced mucosal injury and showed protective effects on the mucosa by reducing the levels of inflammatory cytokines and increasing the expression of antioxidant enzymes and growth factors. Furthermore, PZ showed cytoprotective effects by increasing the HSP levels.

Microglia are a type of resident macrophage that functions as an inflammation modulator in the central nervous system. Over-activation of microglia by a range of stimuli disrupts the physiological homeostasis of the brain, and induces inflammatory response and degenerative processes, such as those implicated in neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Therefore, we investigated the possible anti-inflammatory mechanisms of inflexanin B in murine microglial BV2 cells. Lipopolysaccharide (LPS) activated BV2 cells and induced the production of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), and cytokines (interleukins-1β and -6, and tumour necrosis factor α). The LPS-induced production of pro-inflammatory mediators was associated with the enhancement of nuclear factor-kappaB (NF-κB) nuclear translocation and the activation of mitogen-activated protein kinase (MAPK) including ERK1/2 and JNK. Conversely, pretreatment of cells with inflexanin B (10 and 20 μg/mL) significantly reduced the production of pro-inflammatory mediators. This was accompanied with the reduced nuclear translocation of NF-κB and reduced activation of MAPKs. These results suggest that inflexanin B attenuated the LPS-induced inflammatory process by inhibiting the activation of NF-κB and MAPKs.

Functional amyloid fibers termed curli contribute to bacterial adhesion and biofilm formation in Escherichia coli . We discovered that the nonionic surfactant Tween 20 inhibits biofilm formation by uropathogenic E. coli at the air-liquid interface, referred to as pellicle formation, and at the solid-liquid interface. At Tween 20 concentrations near and above the critical micelle concentration, the interfacial viscoelastic modulus is reduced to zero as cellular aggregates at the air-liquid interface are locally disconnected and eventually eliminated. Tween 20 does not inhibit the production of curli but prevents curli-integrated film formation. Our results support a model in which the hydrophobic curli fibers associated with bacteria near the air-liquid interface require access to the gas phase to formed strong physical entanglements and to form a network that can support shear stress.

Escherichia coli assemble functional amyloid fibers termed curli that contribute to bacterial adhesion, biofilm formation, and host pathogenesis. We developed a cell-based high-throughput screen to identify inhibitors of curli-mediated adhesion in the laboratory strain MC4100 and curli-associated biofilm formation in the uropathogenic E. coli clinical isolate UTI89. Inhibitors of biofilm formation can operate through many mechanisms, and such inhibitors could hold therapeutic value in preventing and treating urinary tract infections. The curli-specific screen allows the identification of compounds that inhibit either curli expression, curli biogenesis, or adhesion by normally produced curli. In screening the NIH Clinical Collection of 446 compounds, we identified rifapentine as a potent inhibitor in both of these screens. Rifapentine is an antibiotic used to treat tuberculosis that targets RNA polymerase, but prevents curli-dependent adhesion and biofilm formation in E. coli at concentrations below those that affect viability. Rifapentine inhibits curli production and prevents biofilm formation on plastic, on agar, and at the air-liquid interface by inhibiting curli gene transcription. Comparisons with a cephalosporin antibiotic further revealed that curli production is not affected by standard antibiotic treatment and cell killing pressure. Thus, we reveal a new role independent of killing activity for rifapentine as an inhibitor of curli and curli-mediated biofilm formation.

Neuroinflammation is chronic inflammation within the brain that is attributed to prolonged activation of microglial cells and results in neurodegenerative events, such as neuronal dysfunction and neuronal loss. Therefore, suppression of neuroinflammation would theoretically slow progression of neurodegenerative disease. In this study, we investigated the anti-inflammatory effects of 4′-O-methylalpinumisoflavone (methylalpinumisoflavone), isolated from Cudrania tricuspidata, against LPS-induced microglial activation in BV2 cells. Exposure of BV2 cells to LPS (0.5 µg/mL) significantly increased production of pro-inflammatory mediators, including NO, PGE2, and pro-inflammatory cytokines. Conversely, pre-treatment with methylalpinumisoflavone (10 and 20 µg/mL) prior to treatment with LPS resulted in a significant decrease of LPS-induced production of pro-inflammatory mediators in a dose-dependent manner. In addition, reduction of pro-inflammatory mediators by treatment with methylalpinumisoflavone prior to treatment with LPS was accompanied by a decrease in translocation of NF-κB p50 and p65 from the cytoplasm to the nucleus and by a decrease in activation of mitogen-activated protein kinases (MAPKs), such as ERK1/2 and JNK. Taken together, these results suggest that methylalpinumisoflavone suppressed LPS-induced microglial activation and production of pro-inflammatory mediators by decreasing NF-κB signaling and by phosphorylation of MAPKs. These results suggest the potential of methylalpinumisoflavone as an anti-inflammatory drug candidate. Copyright © 2012 John Wiley & Sons, Ltd.

Bacterial biofilms are complex multicellular assemblies, characterized by a heterogeneous extracellular polymeric matrix, that have emerged as hallmarks of persistent infectious diseases. New approaches and quantitative data are needed to elucidate the composition and architecture of biofilms, and such data need to be correlated with mechanical and physicochemical properties that relate to function. We performed a panel of interfacial rheological measurements during biofilm formation at the air-liquid interface by the Escherichia coli strain UTI89, which is noted for its importance in studies of urinary tract infection and for its assembly of functional amyloid fibers termed curli. Brewster-angle microscopy and measurements of the surface elasticity (Gs′) and stress-strain response provided sensitive and quantitative parameters that revealed distinct stages during bacterial colonization, aggregation, and eventual formation of a pellicle at the air-liquid interface. Pellicles that formed under conditions that upregulate curli production exhibited an increase in strength and viscoelastic properties as well as a greater ability to recover from stress-strain perturbation. The results suggest that curli, as hydrophobic extracellular amyloid fibers, enhance the strength, viscoelasticity, and resistance to strain of E. coli biofilms formed at the air-liquid interface.

As worldwide efforts to reduce global warming gases, the construction Industry is endeavoring to diminish carbon dioxides emissions. Especially, by introducing the LCA methodology to the industry, A variety of related studies to measure the emission of carbon dioxides have been conducted. However, when the conventional LCA methodology is applied to the construction projects, some limitations have been reported. To overcome the restrictions derived from the industry characteristics, this research suggested the Activity-based LCA model by applying the Activity-based Costing (ABC), which breaks down the whole life cycles into more detailed stages. By implementing the newly developed model, forecasting accuracy of emission was elevated, and the critical control points on carbon dioxides were established. Through the case study of aluminium curtain-wall system, this research verified the usefulness of the Activity-based LCA.

Escherichia coli directs the assembly of functional amyloid fibers termed “curli” that mediate adhesion and biofilm formation. We discovered that E. coli exhibits a tunable and selective increase in curli protein expression and fiber assembly in response to moderate concentrations of dimethyl sulfoxide (DMSO) and ethanol. Furthermore, the molecular alterations resulted in dramatic functional phenotypes associated with community behavior, including (i) cellular agglutination in broth, (ii) altered colony morphology, and (iii) increased biofilm formation. Solid-state nuclear magnetic resonance (NMR) spectra of intact pellicles formed in the presence of [ 13 C 2 ]DMSO confirmed that DMSO was not being transformed and utilized directly for metabolism. Collectively, the chemically induced phenotypes emphasize the plasticity of E. coli 's response to environmental stimuli to enhance amyloid production and amyloid-integrated biofilm formation. The data also support our developing model of the extracellular matrix as an organized assembly of polymeric components, including amyloid fibers, in which composition relates to bacterial physiology and community function.

Using a proteomic approach, a study was conducted for determination of the effects of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PCDF) on proteins secreted by HepG2 cells. Briefly, HepG2 cells were exposed to various concentrations of 2,3,4,7,8-PCDF for 24 or 48 h. MTT and comet assays were then conducted for determination of cytotoxicity and genotoxicity, respectively. Results of an MTT assay showed that 1 nM of 2,3,4,7,8-PCDF was the maximum concentration that did not cause cell death. In addition, a dose- and time dependent increase of DNA damage was observed in HepG2 cells exposed to 2,3,4,7,8-PCDF. Therefore, two different concentrations of 2,3,4,7,8-PCDF, 1 and 5 nM, were selected for further analysis of proteomic biomarkers using two different pI ranges (4–7 and 6–9) and large two dimensional gel electrophoresis. Results showed identification of 32 proteins ( 29 up- and 3 down-regulated) by nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS. Among these, the identities of pyridoxine-5'-phosphate oxidase, UDP-glucose 6-dehydrogenase, plasminogen activator inhibitor I precursor, plasminogen activator inhibitor-3, proteasome activator complex subunit 1, isoform 1 of 14-3-3 protein sigma, peptidyl-prolyl cis-trans isomerase A, 14-3-3 protein gamma, protein DJ-1, and nucleoside diphosphate kinase A were confirmed by western blot analysis. The differential expression of protein DJ-1, proteasome activator complex subunit 1 and plasminogen activator inhibitor-3 was further validated in plasma proteins from rats exposed to 2,3,4,7,8-PCDF. These proteins could be used as potential toxicological biomarkers of 2,3,4,7,8-PCDF.

Objectives We studied several acute inflammatory materials (AIM) such as various inflammatory cytokines, oxidative stress, and heat shock proteins in ARDS patients by simultaneously measuring from bronchoalveolar lavage fluid (BALF) and plasma. Design and methods AIM were measured by using plasma and BALF sampling obtained from ARDS group (n = 12) and non-ARDS group (n = 12). Results In the BALF, only HSP 47 was significantly increased in ARDS group than non-ARDS group. In plasma, GRP 94, HSP 90, HSP 60, HSP 47, GPx-3, and IL-8 were increased significantly in ARDS group. In short, most of the AIM in BALF or plasma were not significantly different in ARDS group as compared with non-ARDS group. Ninety-day mortality was significantly related to HSP90, HSP 60 and GPx-3 in plasma but not in BALF. Conclusion Alteration of AIM levels in both BALF or plasma of ARDS group was not remarkable compared with the non-ARDS group. Our result suggests the need to reconsider ARDS pathophysiology and therapeutic strategy.

The acute administration of ethanol to intestinal epithelial cells causes increased intestinal permeability and the translocation of endotoxins. The changes caused by ethanol in intestinal cells may be related to oxidative stress and DNA damage. However, DNA damage and repair-related molecules which act against stresses, including ethanol, have not been fully investigated in intestinal cells. Heat shock proteins (Hsps) are involved in the recovery and protection from cell damage and may be associated with DNA repair. Therefore, the aim of our study was to investigate cytotoxicity, DNA damage and the expression of DNA repair-related molecules, antioxidant proteins and Hsps in intestinal cells exposed to ethanol. Human intestinal Caco-2 cells were incubated with 1–8% ethanol for 1 h. Cell viability and DNA damage were determined using the MTT and comet assays, respectively. We measured DNA repair-related molecules, including DNA polymerase β, apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1), growth arrest and DNA damage 45α (GADD45α) and proliferating cell nuclear antigen (PCNA), in Caco-2 cells using western blot analysis. We also measured glutathione peroxidase-1 (GPx-1), peroxiredoxin-1 (PRX-1), superoxide dismutase-2 (SOD-2), Hsp10, Hsp27, Hsp60, heat shock cognate (Hsc)70, Hsp70 and Hsp90. The viability of the Caco-2 cells exposed to ethanol decreased at concentrations ≥7% (P

Neurovascular degeneration contributes to the pathogenesis of Alzheimer's disease (AD). Because erythropoietin (EPO) promotes endothelial regeneration, we investigated the therapeutic effects of EPO in animal models of AD. In aged Tg2576 mice, EPO receptors (EPORs) were expressed in the cortex and hippocampus. Tg2576 mice were treated with daily injection of EPO (5000 IU/kg/day) for 5 days. At 14 days, EPO improved contextual memory as measured by fear-conditioning test. EPO enhanced endothelial proliferation and the level of synaptophysin expression in the brain. EPO also increased capillary density, and decreased the level of the receptor for advanced glycation endproducts (RAGE) in the brain, while decreasing in the amount of amyloid plaque and amyloid-β (Aβ). In cultured human endothelial cells, EPO enhanced angiogenesis and suppressed the expression of the RAGE. These results show that EPO improves memory and ameliorates endothelial degeneration induced by Aβ in AD models. This pre-clinical evidence suggests that EPO may be useful for the treatment of AD.

Proteomic changes in proteins secreted by human hepatocellular carcinomas (HepG2) cells exposed to butyl benzyl phthalate (BBP) were evaluated. HepG2 cells were treated with three different concentrations of BBP (0, 10, or 25 μM) for 24 or 48 h. Following incubation, the cells were subjected to proteomic analysis using two different pI ranges (4-7 and 6-9) and large-size two-dimensional gel electrophoresis. Results showed resolution of a total of 2776 protein spots. Of these, 29, including 19 upregulated and 10 downregulated proteins, were identified by electrospray ionization-mass spectrometry-mass spectrometry (ESI-MS/MS). Among these, the identities of cystatin C, Rho guanine nucleotide dissociation inhibitor, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, heptaglobin-related protein, inter-alpha-trypsin inhibitor heavy chain H2, and electron transfer flavoprotein subunit beta were confirmed by Western blot analysis. These proteins were found to be involved in apoptosis, signaling, tumor progression, energy metabolism, and cell structure and motility. Therefore, these proteins have potential to be employed as biomarkers of BBP exposure and may be useful in understanding mechanisms underlying the adverse effects of BBP.

Neuroinflammation with prolonged microglial activation leads to increased levels of pro-inflammatory mediators and subsequently contributes to neuronal dysfunction and neuronal loss. Therefore, pharmacological suppression of neuroinflammation would theoretically slow the progression of neurodegenerative disease. In this study, we investigated the anti-inflammatory effects and possible mechanisms of isodojaponin D (19-hydroxy-1alpha,6-diacetoxy-6,7-seco-ent-kaur-16-en-15-one-7,20-olide), a new diterpene isolated from Isodon japonicus against lipopolysaccharide(LPS)-induced microglial activation in BV2 cells. Results from RT-PCR and Western blot showed that pretreatment with isodojaponin D (5 and 10 microg/ml) prior to treatment with LPS (1 microg/ml) significantly decreased LPS-induced production of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in a dose-dependent manner. In addition, LPS-induced pro-inflammatory cytokines, including IL-1beta, IL-6, and TNF-alpha, were also decreased by pretreatment with isodojaponin D. This effect was accompanied by a decrease in translocations of Nuclear Factor-KappaB (NF-kappaB) p50 and p65 from the cytoplasm to the nucleus and by a decrease in I kappaB (IkappaB) degradation. In addition, pretreatment with isodojaponin D significantly attenuated LPS-induced mitogen-activated protein kinase (MAPK) activation. Taken together, these results suggest that isodojaponin D suppressed LPS-induced microglial activation and production of pro-inflammatory mediators by inhibition of the NF-kappaB signaling pathway and phosphorylation of MAPKs. These results suggest that isodojaponin D could play a beneficial role in treatment of neurodegenerative disease.

Escherichia coli O157:H7, a food-borne pathogen, causes hemorrhagic colitis and the hemolytic-uremic syndrome. A putative virulence factor of E. coli O157:H7 is a 60-MDa plasmid (pO157) found in 99% of all clinical isolates and many bovine-derived strains. The well characterized E. coli O157:H7 Sakai strain (Sakai) and its pO157-cured derivative (Sakai-Cu) were compared for phenotypic differences. Sakai-Cu had enhanced survival in synthetic gastric fluid, did not colonize cattle as well as wild-type Sakai, and had unchanged growth rates and tolerance to salt and heat. These results are consistent with our previous findings with another E. coli O157:H7 disease outbreak isolate ATCC 43894 and its pO157-cured (43894-Cu). However, despite the essentially sequence identical pO157 in these strains, Sakai-Cu had changes in antibiotic susceptibility and motility that did not occur in the 43894-Cu strain. This unexpected result was systematically analyzed using phenotypic microarrays testing 1,920 conditions with Sakai, 43894, and the plasmid-cured mutants. The influence of the pO157 differed between strains on a wide number of growth/survival conditions. Relative expression of genes related to acid resistance (gadA, gadX, and rpoS) and flagella production (fliC and flhD) were tested using quantitative real-time PCR and gadA and rpoS expression differed between Sakai-Cu and 43894-Cu. The strain-specific differences in phenotype that resulted from the loss of essentially DNA-sequence identical pO157 were likely due to the chromosomal genetic diversity between strains. The O157:H7 serotype diversity was further highlighted by phenotypic microarray comparisons of the two outbreak strains with a genotype 6 bovine E. coli O157:H7 isolate, rarely associated with human disease.

Construction Projects have mutual collaborative structures among various participants for common objectives. Most of all the design project which is the initial phase in the project is crucial for determining project overall quality and requires intimate and continuous relations between project participants. Thus, this study suggests Design Collaboration Management System for successful performing projects through cooperation among participants in the initial stage of projects. First, expert interviews were performed to figure out present conditions of design collaboration management and problems. The factor analysis was applied to draw owner`s collaboration factors and engineer`s collaboration factors. Based on those factors, this study suggests design cooperation management process which gives a framework for systematic management method. Through the process the design collaboration management system was developed which helps owner/designers/engineer`s co-work. The System suggested from this study realizes systematic information management tool and through that it is expected to improve design product;`s quality, and further contribute the advancement of project.

The role of plasmid pO157 in biofilm formation was investigated using wild-type and pO157-cured Escherichia coli O157:H7 Sakai. Compared to the wild type, the biofilm formed by the pO157-cured mutant produced fewer extracellular carbohydrates, had lower viscosity, and did not give rise to colony morphology variants that hyperadhered to solid surfaces.

Enterohemorrhagic Escherichia coli O157:H7 is a major foodborne pathogen causing severe disease in humans worldwide. Healthy cattle are a reservoir of E. coli O157:H7, and bovine food products and fresh produce contaminated with bovine waste are the most common sources for disease outbreaks in the United States. E. coli O157:H7 also survives well in the environment. The abilities to cause human disease, colonize the bovine gastrointestinal tract, and survive in the environment require that E. coli O157:H7 adapt to a wide variety of conditions. Three major virulence factors of E. coli O157:H7 have been identified including Shiga toxins, products of the pathogenicity island called the locus of enterocyte effacement, and products of the F-like plasmid pO157. Among these virulence factors, the role of pO157 is least understood. This review provides a board overview of E. coli O157:H7 with an emphasis on pO157.

Alzheimer's disease is the most common form of dementia, causing progressive cognitive dysfunction, particularly memory loss. Recently, modulation of beta-amyloid (Abeta) toxicity, one of the major potential causes of Alzheimer's disease, has emerged as a possible therapeutic approach to control the onset of Alzheimer's disease. In this study, we investigated the neuroprotective effects and possible mechanisms by which 19-hydroxy-1alpha,6-diacetoxy-6,7-seco-ent-kaur-16-en-15-one-7,20-olide (named as CBNU06), a new diterpene isolated from Isodon japonicus, acts against Abeta-induced toxicity in PC12 cells. Pretreatment with CBNU06 (20 microg/ml) prior to Abeta(25)(-35) (25 microM) significantly increased the viability of PC12 cells in a dose-dependent manner when examined by Hoechst staining, MTT assay and Trypan blue exclusion assay. This protective effect was accompanied by the decrease in translocation of NF-kappaB p50 and p65 from the cytoplasm to the nucleus, and followed by the decrease in cyclooxygenase-2 (COX-2) levels. In addition, pretreatment with CBNU06 significantly reversed the effect of Abeta on Bax and Bcl-2. Taken together, these results suggest that CBNU06 protected PC12 cells against Abeta-induced neurotoxicity through the inhibition of the NF-kappaB signaling pathways. Therefore, CBNU06 has the possible beneficial effects in Alzheimer's disease by attenuating Abeta-induced toxicity.

Toxicological biomarkers of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) were investigated in proteins secreted by HepG2 cells and their expression levels were determined in the plasma of rats exposed to 2,3,7,8-TCDD and in the plasma of incineration workers exposed to dioxins. HepG2 cells were treated with various concentrations of 2,3,7,8-TCDD (0, 0.25, 0.5, 1, 2.5, 5, 10, 25 nM) for 24 or 48 h. MTT and Comet assays were performed to determine cytotoxicities and genotoxicities to select exposure concentrations for the proteomic analysis of proteins secreted by 2,3,7,8-TCDD-treated cells. In the proteomic analysis, dose- and time-dependent toxicological biomarkers were evaluated using two pI ranges (4-7 and 6-9) using a large gel 2-DE system. Fifteen secreted proteins were identified by a nano-LC-ESI-MS/MS and nano-ESI on a Q-TOF2 MS and the identities of eight secreted proteins including glyoxalase 1 (GLO 1), homogentisate dioxygenase (HGD), peroxiredoxin 1 (PRX 1), proteasome subunit beta type (PSMB) 5 and 6, UDP-glucose 6-dehydrogenase (UDP-GlcDH), hydroxyacyl-coenzyme A dehydrogenase (HADH) and serotransferrin (STF) were confirmed by western blotting. Of these, PSMB 5 and PRX 1 were also found in the plasma of rats exposed to 2,3,7,8-TCDD, whereas GLO 1, HGD, PSMB 6 and PRX 1 were found in the plasma of incineration workers exposed to dioxins.

The human pathogen Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening sequelae and transiently colonizes healthy cattle at the terminal rectal mucosa. This study analyzed virulence factors important for the clinical manifestations of human E. coli O157:H7 infection for their contribution to the persistence of E. coli in cattle. The colonizing ability of E. coli O157:H7 was compared with those of nonpathogenic E. coli K-12 and isogenic deletion mutants missing Shiga toxin (Stx), the adhesin intimin, its receptor Tir, hemolysin, or the ∼92-kb pO157. Fully ruminant steers received a single rectal application of one E. coli strain so that effects of mucosal attachment and survival at the terminal rectum could be measured without the impact of bacterial passage through the entire gastrointestinal tract. Colonization was monitored by sensitive recto-anal junction mucosal swab culture. Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. The E. coli O157:H7 best able to persist had intimin, Tir, and the pO157. Strains missing even one of these factors were recovered in lower numbers and were cleared faster than the wild type. In contrast, E. coli O157:H7 strains that were missing Stx or hemolysin colonized like the wild type. For these three strains, the number of bacteria increased between days 1 and 4 postapplication and then decreased slowly. In contrast, the numbers of noncolonizing strains (K-12, Δ tir , and Δ eae ) decreased from the day of application. These patterns consistently predicted long-term colonization or clearance of the bacteria from the bovine terminal rectal mucosa.

Vancomycin resistant enterococci (VRE) isolates from humans (23 isolates) and poultry (20 isolates) were characterized by antibiotic susceptibility, vancomycin resistance transferability, pulsed-field gel electrophoresis (PFGE), and structural analysis of Tn1546-like elements. VRE isolates from humans and poultry showed different resistance patterns, transferability, and transfer rate. In addition to these phenotypic differences between humans and poultry VRE, PFGE and the structure of Tn1546-like elements were also distinct. Most poultry isolates (16/20) were identical to the prototype vanA transposon, Tn1546, while most human isolates (21/23) had multiple integrations of insertion sequence. The transmission of VRE and vancomycin resistance determinant between humans and poultry could not be demonstrated in this study.

Escherichia coli O157:H7 is an important food-borne pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome in humans. Recently, we reported that the pO157 ecf ( E. coli attaching and effacing gene-positive conserved fragments) operon is thermoregulated by an intrinsically curved DNA and contains the genes for bacterial surface-associated proteins, including a second copy of lipid A myristoyl transferase, whose chromosomal copy is the lpxM gene product. E. coli O157:H7 survives and persists well in diverse environments from the human and bovine gastrointestinal tracts (GIT) to nutrient-dilute farm water troughs. Transcriptional regulation of the ecf operon by intrinsic DNA curvature and the genetic redundancy of lpxM that is associated with lipid A modification led us to hypothesize that the pO157 ecf operon and lpxM are associated with bacterial survival and persistence in various in vivo and ex vivo environments by optimizing bacterial membrane structure and/or integrity. To test this hypothesis, three isogenic ecf operon and/or lpxM deletion mutants of E. coli O157:H7 ATCC 43894 were constructed and analyzed in vitro and in vivo. The results showed that a double mutant carrying deletions in the ecf and lpxM genes had an altered lipid A structure and membrane fatty acid composition, did not survive passage through the bovine GIT, did not persist well in farm water troughs, had increased susceptibility to a broad spectrum of antibiotics and detergents, and had impaired motility. Electron microscopic analyses showed gross changes in bacterial membrane structure.

We have shown that long-term intake of Korean red ginseng (KRG) delays disease progression in HIV-1 infected patients. In the present study to investigate whether this slow progression was associated with protective human leukocyte antigen (HLA) alleles as well as with KRG-intake, we have performed clinical analysis of 31 HIV-1 infected patients who have been living for more than 10 years without any antiretroviral therapy. Average amount of KRG-intake over 130±16 months was 4,797±4,921 g and the annual decrease in CD4 T cell (AD) was 30±29/㎕. We observed significant correlations among amount of KRG-intake, AD (r = -0.53, P < 0.01), and plasma HIV-1 RNA copy (r = -0.35, P < 0.05), along with a significant correlation between KRG-intake and HLA score (r = 0.49, P < 0.01), whereas there was no significant correlation between HLA score and AD or viral load. When the 31 patients were divided into 2 groups based on the amount of KRG-intake, the AD (14/㎕) in the 16 patients who had taken higher amounts ofKRG was significantly less than that (49/㎕) in the 15 patients with a little or no KRG-intake (P < 0.01). These data indicate that KRG-intake significantly slows CD4 T cell depletion in HIV-1 infected patients.

Long-duration consistently Escherichia coli O157:H7 culture-positive cattle were euthanized and necropsied. Tissue and digesta from along the gastrointestinal tract (GIT) were cultured for the bacteria and examined histologically for lymphoid character. E. coli O157:H7 was detected only at the rectoanal junction mucosa and not at any other GIT location.

This study profiled the plasma proteins of patients infected by the 2011 H1N1 influenza virus. Differential protein expression was identified in plasma obtained from noninfected control subjects (n = 15) and H1N1-infected subjects (n = 15). Plasma proteins were separated by a 2DE large gel system and identified by nano-ultra performance LC-MS. Western blot assays were performed to validate proteins. Eight plasma proteins were upregulated and six proteins were downregulated among 3316 plasma proteins in the H1N1-infected group as compared with the control group. Of 14 up- and downregulated proteins, nine plasma proteins were validated by Western blot analysis. Putative protein FAM 157A, leucine-rich alpha 2 glycoprotein, serum amyloid A protein, and dual oxidase 1 showed significant differential expression. The identified plasma proteins could be potential candidates for biomarkers of H1N1 influenza viral infection. Further studies are needed to develop these proteins as diagnostic biomarkers.

Biofilms are multi-cellular communities formed by bacteria, and they consist of bacteria encased within a non-crystalline extracellular matrix (ECM) of proteins, polysaccharides, and small molecules. Biofilm formation provides increased protection of bacteria from antibiotics and host defenses. New quantitative approaches are needed to define composition and architecture of biofilms. We have utilized solid-state NMR to quantitatively define the composition of the wild-type uropathogenic E. coli UTI89 extracellular matrix. Recently, we have extended the same methodology to elucidate changes in biofilm composition due to inhibitory small molecules and to define the composition of Vibrio cholerae biofilm.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Uropathogenic Escherichia coli (UPEC) are the major causative agents of urinary tract infection and engage in a coordinated genetic and molecular cascade to colonize the urinary tract. Disrupting the assembly and/or function of virulence factors and bacterial biofilms has emerged as an attractive target for the development of new therapeutic strategies to prevent and treat urinary tract infection, particularly in the era of increasing antibiotic resistance among human pathogens. UPEC vary widely in their genetic and molecular phenotypes and more data are needed to understand the features that distinguish isolates as more or less virulent and as more robust biofilm formers or poor biofilm formers. Curli are extracellular functional amyloid fibers produced by E. coli that contribute to pathogenesis and influence the host response during urinary tract infection (UTI). We have examined the production of curli and curli-associated phenotypes including biofilm formation among a specific panel of human clinical UPEC that has been studied extensively in the mouse model of UTI. Motility, curli production, and curli-associated biofilm formation attached to plastic were the most prevalent behaviors, shared by most clinical isolates. We discuss these results in the context on the previously reported behavior and phenotypes of these isolates in the murine cystitis model in vivo.

A new sample-to-answer polymer lab-on-a-chip, which can perform immunoassay with minimum user intervention through on-chip reservoirs for reagents and single-channel assay system, has been designed, developed and successfully characterized as a point-of-care testing (POCT) cartridge for the detection of thyroid stimulating hormone (TSH). Test results were obtained within 30 minutes after a sample was dropped into the POCT cartridge. The analyzed results of TSH showed a linear range of up to 55 μIU mL(-1) with the limit of detection (LOD) of 1.9 μIU mL(-1) at the signal-to-noise ratio (SNR) of 3. The reagents stored in the on-chip reservoirs maintained more than 97% of their initial volume for 120 days of storage time while the detection antibody retained its activity above 98% for 120 days. The sample-to-answer polymer lab-on-a-chip developed in this work using the mass-producible and low-cost polymer is well suited for the point-of-care testing of rapid in vitro diagnostics (IVD) of TSH.

Neurovascular degeneration contributes to the pathogenesis of Alzheimer's disease (AD). Because erythropoietin (EPO) promotes endothelial regeneration, we investigated the therapeutic effects of EPO in animal models of AD. In aged Tg2576 mice, EPO receptors (EPORs) were expressed in the cortex and hippocampus. Tg2576 mice were treated with daily injection of EPO (5000 IU/kg/day) for 5 days. At 14 days, EPO improved contextual memory as measured by fear-conditioning test. EPO enhanced endothelial proliferation and the level of synaptophysin expression in the brain. EPO also increased capillary density, and decreased the level of the receptor for advanced glycation endproducts (RAGE) in the brain, while decreasing in the amount of amyloid plaque and amyloid-β (Aβ). In cultured human endothelial cells, EPO enhanced angiogenesis and suppressed the expression of the RAGE. These results show that EPO improves memory and ameliorates endothelial degeneration induced by Aβ in AD models. This pre-clinical evidence suggests that EPO may be useful for the treatment of AD.

Neuroinflammation is chronic inflammation within the brain that is attributed to prolonged activation of microglial cells and results in neurodegenerative events, such as neuronal dysfunction and neuronal loss. Therefore, suppression of neuroinflammation would theoretically slow progression of neurodegenerative disease. In this study, we investigated the anti-inflammatory effects of 4'-O-methylalpinumisoflavone (methylalpinumisoflavone), isolated from Cudrania tricuspidata, against LPS-induced microglial activation in BV2 cells. Exposure of BV2 cells to LPS (0.5 µg/mL) significantly increased production of pro-inflammatory mediators, including NO, PGE(2), and pro-inflammatory cytokines. Conversely, pre-treatment with methylalpinumisoflavone (10 and 20 µg/mL) prior to treatment with LPS resulted in a significant decrease of LPS-induced production of pro-inflammatory mediators in a dose-dependent manner. In addition, reduction of pro-inflammatory mediators by treatment with methylalpinumisoflavone prior to treatment with LPS was accompanied by a decrease in translocation of NF-κB p50 and p65 from the cytoplasm to the nucleus and by a decrease in activation of mitogen-activated protein kinases (MAPKs), such as ERK1/2 and JNK. Taken together, these results suggest that methylalpinumisoflavone suppressed LPS-induced microglial activation and production of pro-inflammatory mediators by decreasing NF-κB signaling and by phosphorylation of MAPKs. These results suggest the potential of methylalpinumisoflavone as an anti-inflammatory drug candidate.