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Find video protocols related to scientific articles indexed in Pubmed.
Flavobacterium suzhouense sp. nov., isolated from farmland river sludge.
Int. J. Syst. Evol. Microbiol.
PUBLISHED: 11-02-2014
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A Gram-negative bacterium, designated XIN-1T, was isolated from farmland river sludge sample in Suzhou, China. Cells of strain XIN-1T was strictly aerobic, non-motile and rod-shaped. Strain XIN-1T grew optimally at pH 7.0 and 28?. Phylogenetic analysis of the 16S rRNA gene sequences showed that strain XIN-1T was most closely related to Flavobacterium hauense BX12T (98.2 %), followed by Flavobacterium beibuense F44-8T (96.3 %). The major respiratory quinone was menaquinone-6 and the major polar lipid was phosphatidylethanolamine. The major fatty acids (?5 %) were summed feature 3 (comprising C16:1?7c and/or C16:1?6c), summed feature 4 (comprising C17:1iso I and/or C17:1anteiso B), iso-C15:0, C16:0 and iso-C17:0 3-OH. The genomic DNA G+C content of strain XIN-1T was 39.8 mol %. Strain XIN-1T showed low DNA-DNA relatedness with F.hauense BX12T (38.7±0.5 %). On the basis of genotypic and phenotypic data, strain XIN-1T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium suzhouense sp. nov. is proposed. The type strain is XIN-1T (=CCTCC AB 2014200T =KCTC 42107T).
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Cloning, expression and characterisation of a type II cystatin from Schistosoma japonicum, which could regulate macrophage activation.
Parasitol. Res.
PUBLISHED: 06-14-2014
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Cystatin play an important role in parasite immune evasion. It is involved in many immune responses processes regulations such as inhibiting antigen presentation, modifying cytokines production and macrophage polarization. In recent years, more and more cystatins were used in treating some inflammatory diseases such as asthma and inflammation bowel diseases; however, cystatins from Schistosoma japonicum were rarely studied. In the present study, we have cloned a cystatin from the adult stage of Schistosoma japonicum, named as SjCystatin, and its sequence shares conserved domains with other type II family cystatins. It was further verified by enzyme inhibition assays. SjCystatin retained its inhibitory activity under a wide range of pH values and temperatures, can maintain its inhibitory activity at pH 6.5-7.5 and 37 °C, respectively. Then, we investigated the effects of SjCystatin on the lipopolysaccharide (LPS)-induced activated RAW264.7. Results showed that SjCystatin inhibit LPS-induced nitric oxide production in a dose-dependent manner. LPS-induced TNF-? and IL-6 production began to be inhibited at least 6 h after SjCystatin stimulation. SjCystatin significantly increased IL-10 production at 6 h after stimulation and its effect on IL-10 production diminished quickly. These results imply that SjCystatin can induce M2 macrophage polarization and can be expected to serve as a potential drug source for the medication of inflammatory disorders like other cystatins.
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Intranasal adenovirus-vectored vaccine for induction of long-lasting humoral immunity-mediated broad protection against influenza in mice.
J. Virol.
PUBLISHED: 06-11-2014
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Influenza vaccines aimed at inducing antibody (Ab) responses against viral surface hemagglutinin (HA) and neuraminidase (NA) provide sterile immunity to infection with the same subtypes. Vaccines targeting viral conserved determinants shared by the influenza A viruses (IAV) offer heterosubtypic immunity (HSI), a broad protection against different subtypes. We proposed that vaccines targeting both HA and the conserved ectodomain of matrix protein 2 (M2e) would provide protection against infection with the same subtype and also HSI against other subtypes. We report here that single intranasal immunization with a recombinant adenovirus (rAd) vector encoding both HA of H5 virus and M2e (rAdH5/M2e) induced significant HA- and M2e-specific Ab responses, along with protection against heterosubtypic challenge in mice. The protection is superior compared to that induced by rAd vector encoding either HA (rAdH5), or M2e (rAdM2e). While protection against homotypic H5 virus is primarily mediated by virus-neutralizing Abs, the cross-protection is associated with Abs directed to conserved stalk HA and M2e that seem to have an additive effect. Consistently, adoptive transfer of antisera induced by rAdH5/M2e provided the best protection against heterosubtypic challenge compared to that provided by antisera derived from mice immunized with rAdH5 or rAdM2e. These results support the development of rAd-vectored vaccines encoding both H5 and M2e as universal vaccines against different IAV subtypes.
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Vaccinia-based influenza vaccine overcomes previously induced immunodominance hierarchy for heterosubtypic protection.
Eur. J. Immunol.
PUBLISHED: 03-21-2014
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Growing concerns about unpredictable influenza pandemics require a broadly protective vaccine against diverse influenza strains. One of the promising approaches was a T cell-based vaccine, but the narrow breadth of T-cell immunity due to the immunodominance hierarchy established by previous influenza infection and efficacy against only mild challenge condition are important hurdles to overcome. To model T-cell immunodominance hierarchy in humans in an experimental setting, influenza-primed C57BL/6 mice were chosen and boosted with a mixture of vaccinia recombinants, individually expressing consensus sequences from avian, swine, and human isolates of influenza internal proteins. As determined by IFN-? ELISPOT and polyfunctional cytokine secretion, the vaccinia recombinants of influenza expanded the breadth of T-cell responses to include subdominant and even minor epitopes. Vaccine groups were successfully protected against 100 LD50 challenges with PR/8/34 and highly pathogenic avian influenza H5N1, which contained the identical dominant NP366 epitope. Interestingly, in challenge with pandemic A/Cal/04/2009 containing mutations in the dominant epitope, only the group vaccinated with rVV-NP + PA showed improved protection. Taken together, a vaccinia-based influenza vaccine expressing conserved internal proteins improved the breadth of influenza-specific T-cell immunity and provided heterosubtypic protection against immunologically close as well as distant influenza strains.
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Evaluation of heterosubtypic cross-protection against highly pathogenic H5N1 by active infection with human seasonal influenza A virus or trivalent inactivated vaccine immunization in ferret models.
J. Gen. Virol.
PUBLISHED: 01-13-2014
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The threat of highly pathogenic avian influenza (HPAI) H5N1 viruses to cause the next pandemic remains a major concern. Here, we evaluated the cross-protection induced by natural infection of human seasonal influenza strains or immunization with trivalent inactivated influenza vaccine (TIV) against HPAI H5N1 (A/Vietnam/1203/2004) virus in ferrets. Groups were treated with PBS (group A), infected with H1N1 (group B) or H3N2 (group C) virus, or immunized with TIV (group D). Twelve weeks after the last treatment, serological assays revealed that groups B and C, but not group D, sustained moderate immunogenicity against homologous viruses; cross-reactivity against the H5N1 virus was not detected in any group. Following challenge with A/Vietnam/1203/2004 (H5N1) virus, only groups B and C exhibited attenuated viral loads leading to 100?% survival. Our data suggest that natural infection with human seasonal strains could potentially provide better heterosubtypic protection against HPAI H5N1 virus infection compared to TIV immunization.
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Development of safe and effective RSV Vaccine by modified CD4 epitope in G protein core fragment (Gcf).
PLoS ONE
PUBLISHED: 01-01-2014
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Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in infants and young children worldwide, but currently no safe and effective vaccine is available. The RSV G glycoprotein (RSVG), a major attachment protein, is an important target for the induction of protective immune responses during RSV infection. However, it has been thought that a CD4+ T cell epitope (a.a. 183-195) within RSVG is associated with pathogenic pulmonary eosinophilia. To develop safe and effective RSV vaccine using RSV G protein core fragment (Gcf), several Gcf variants resulting from modification to CD4+ T cell epitope were constructed. Mice were immunized with each variant Gcf, and the levels of RSV-specific serum IgG were measured. At day 4 post-challenge with RSV subtype A or B, lung viral titers and pulmonary eosinophilia were determined and changes in body weight were monitored. With wild type Gcf derived from RSV A2 (wtAGcf), although RSV A subtype-specific immune responses were induced, vaccine-enhanced disease characterized by excessive pulmonary eosinophil recruitment and body weight loss were evident, whereas wtGcf from RSV B1 (wtBGcf) induced RSV B subtype-specific immune responses without the signs of vaccine-enhanced disease. Mice immunized with Th-mGcf, a fusion protein consisting CD4+ T cell epitope from RSV F (F51-66) conjugated to mGcf that contains alanine substitutions at a.a. position 185 and 188, showed higher levels of RSV-specific IgG response than mice immunized with mGcf. Both wtAGcf and Th-mGcf provided complete protection against RSV A2 and partial protection against RSV B. Importantly, mice immunized with Th-mGcf did not develop vaccine-enhanced disease following RSV challenge. Immunization of Th-mGcf provided protection against RSV infection without the symptom of vaccine-enhanced disease. Our study provides a novel strategy to develop a safe and effective mucosal RSV vaccine by manipulating the CD4+ T cell epitope within RSV G protein.
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Mucosal vaccination with recombinant Lactobacillus casei-displayed CTA1-conjugated consensus matrix protein-2 (sM2) induces broad protection against divergent influenza subtypes in BALB/c mice.
PLoS ONE
PUBLISHED: 01-01-2014
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To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with (pgsA-CTA1-sM2/L. casei) or without (pgsA-sM2/L. casei) cholera toxin subunit A1 (CTA1) on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD50 of A/EM/Korea/W149/06(H5N1), A/Puerto Rico/8/34(H1N1), A/Aquatic bird /Korea/W81/2005(H5N2), A/Aquatic bird/Korea/W44/2005(H7N3), and A/Chicken/Korea/116/2004(H9N2) viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes.
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Supplementation of oil-based inactivated H9N2 vaccine with M2e antigen enhances resistance against heterologous H9N2 avian influenza virus infection.
Vet. Microbiol.
PUBLISHED: 01-01-2014
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Avian influenza virus (AIV) subtype H9N2 has been evolving rapidly and vaccine escape variants have been reported to cause circulation of infections and economic losses. In the present study, we developed and evaluated ectodomain of the AIV matrix 2 (M2e) protein as a supplementing antigen for oil-based inactivated H9N2 vaccine to increase resistance against vaccine escape variants. AIV H9N2 M2e antigen was expressed in Escherichia coli and supplemented to inactivated H9N2 oil emulsion vaccine. Specific pathogen-free chickens received a single injection of inactivated H9N2 oil emulsion vaccines with or without M2e supplementation. At three weeks post vaccination, hemagglutination inhibition tests and enzyme-linked immunosorbent assays were performed to determine serological immune responses. Challenge study using a vaccine escape H9N2 variant was performed to evaluate the efficacy of M2e supplementation. M2e antigen supplemented in oil emulsion vaccine was highly immunogenic, and a single M2e-supplemented vaccination reduced challenge virus replication and shedding more effectively than non-supplemented vaccination.
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Mucosal immunization with recombinant adenovirus encoding soluble globular head of hemagglutinin protects mice against lethal influenza virus infection.
Immune Netw
PUBLISHED: 12-03-2013
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Influenza virus is one of the major sources of respiratory tract infection. Due to antigenic drift in surface glycoproteins the virus causes annual epidemics with severe morbidity and mortality. Although hemagglutinin (HA) is one of the highly variable surface glycoproteins of the influenza virus, it remains the most attractive target for vaccine development against seasonal influenza infection because antibodies generated against HA provide virus neutralization and subsequent protection against the virus infection. Combination of recombinant adenovirus (rAd) vector-based vaccine and mucosal administration is a promising regimen for safe and effective vaccination against influenza. In this study, we constructed rAd encoding the globular head region of HA from A/Puerto Rico/8/34 virus as vaccine candidate. The rAd vaccine was engineered to express high level of the protein in secreted form. Intranasal or sublingual immunization of mice with the rAd-based vaccine candidates induced significant levels of sustained HA-specific mucosal IgA and IgG. When challenged with lethal dose of homologous virus, the vaccinated mice were completely protected from the infection. The results demonstrate that intranasal or sublingual vaccination with HA-encoding rAd elicits protective immunity against infection with homologous influenza virus. This finding underlines the potential of our recombinant adenovirus-based influenza vaccine candidate for both efficacy and rapid production.
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Neonatal immunization with respiratory syncytial virus glycoprotein fragment induces protective immunity in the presence of maternal antibodies in mice.
Viral Immunol.
PUBLISHED: 07-19-2013
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Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly worldwide. The significant morbidity and mortality associated with this infection underscores the urgent need for development of RSV vaccine. In this study, we first show that intranasal administration of RSV glycoprotein core fragment (Gcf) to neonatal mice can induce systemic humoral immune responses and protective immunity against RSV without causing lung eosinophilia, although antibody response was shifted to a Th2 response. Next, we examined whether the presence of maternal anti-RSV antibodies would affect the responsiveness and protection efficacy of Gcf in newborn mice, since infants can possess RSV-specific maternal antibodies due to frequent RSV re-infections to adults. Intranasal administration of Gcf induced antibody response and increased IFN? secretion and protected mice against RSV challenge without severe lung eosinophilia, even in the presence of high levels of RSV-specific maternal antibodies. Thus, our findings suggest that Gcf may be an effective and safe RSV vaccine during the neonatal period.
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Sublingual delivery of vaccines for the induction of mucosal immunity.
Immune Netw
PUBLISHED: 05-02-2013
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The mucosal surfaces are constantly exposed to incoming pathogens which can cause infections that result in severe morbidity and/or mortality. Studies have reported that mucosal immunity is important for providing protection against these pathogens and that mucosal vaccination is effective in preventing local infections. For many years, the sublingual mucosa has been targeted to deliver immunotherapy to treat allergic hypersensitivities. However, the potential of vaccine delivery via sublingual mucosal has received little attention until recently. Recent studies exploring such potential have documented the safety and effectiveness of sublingual immunization, demonstrating the ability of sublingual immunization to induce both systemic and mucosal immune responses against a variety of antigens, including soluble proteins, inter particulate antigens, and live-attenuated viruses. This review will summarize the recent findings that address the promising potential of sublingual immunization in proving protection against various mucosal pathogens.
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Immunogenicity and safety of H1N1 influenza hemagglutinin protein expressed in a baculovirus system.
Microbiol. Immunol.
PUBLISHED: 03-27-2013
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Although most influenza vaccines are produced in eggs, new types of vaccines must be developed. In this study, the immunogenicity and safety of a baculovirus-expressed hemagglutinin (HA) of H1N1 influenza virus (Korea/01/2009; designated "HA-Bac-K") was compared with those of a commercially available baculovirus-expressed HA (designated "HA-Bac-C") and an Escherichia coli-expressed HA (designated "HA-E. Coli-K"). HA-Bac-K succeeded in inducing hemagglutination inhibition and neutralization antibodies in mouse and ferret models. The different immunogenicities observed may be attributable to the different expression systems and purification protocols used. Our work suggests that HA expressed in a baculovirus system is an effective and safe candidate influenza vaccine.
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Prokaryote-expressed M2e protein improves H9N2 influenza vaccine efficacy and protection against lethal influenza A virus in mice.
Virol. J.
PUBLISHED: 03-22-2013
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Influenza vaccines are prepared annually based on global epidemiological surveillance data. However, since there is no method by which to predict the influenza strain that will cause the next pandemic, the demand to develop new vaccination strategies with broad cross-reactivity against influenza viruses are clearly important. The ectodomain of the influenza M2 protein (M2e) is an attractive target for developing a vaccine with broad cross-reactivity. For these reasons, we investigated the efficacy of an inactivated H9N2 virus vaccine (a-H9N2) mixed with M2e (1xM2e or 4xM2e) proteins expressed in Escherichia coli, which contains the consensus of sequence the extracellular domain of matrix 2 (M2e) of A/chicken/Vietnam/27262/09 (H5N1) avian influenza virus, and investigated its humoral immune response and cross-protection against influenza A viruses.
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Multiple Nudix family proteins possess mRNA decapping activity.
RNA
PUBLISHED: 01-25-2013
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RNA decapping is an important contributor to gene expression and is a critical determinant of mRNA decay. The recent demonstration that mammalian cells harbor at least two distinct decapping enzymes that preferentially modulate a subset of mRNAs raises the intriguing possibility of whether additional decapping enzymes exist. Because both known decapping proteins, Dcp2 and Nudt16, are members of the Nudix hydrolase family, we set out to determine whether other members of this family of proteins also contain intrinsic RNA decapping activity. Here we demonstrate that six additional mouse Nudix proteins--Nudt2, Nudt3, Nudt12, Nudt15, Nudt17, and Nudt19--have varying degrees of decapping activity in vitro on both monomethylated and unmethylated capped RNAs. The decapping products from Nudt17 and Nudt19 were analogous to Dcp2 and predominantly generated m?GDP, while cleavage by Nudt2, Nudt3, Nudt12, and Nudt15 was more pleiotropic and generated both m?GMP and m?GDP. Interestingly, all six Nudix proteins as well as both Dcp2 and Nudt16 could hydrolyze the cap of an unmethylated capped RNA, indicating that decapping enzymes may be less constrained for the presence of the methyl moiety. Investigation of Saccharomyces cerevisiae Nudix proteins revealed that the yeast homolog of Nudt3, Ddp1p, also possesses decapping activity in vitro. Moreover, the bacterial Nudix pyrophosphohydrolase RppH displayed RNA decapping activity and released m?GDP product comparable to Dcp2, indicating that decapping is an evolutionarily conserved activity that preceded mammalian cap formation. These findings demonstrate that multiple Nudix family hydrolases may function in mRNA decapping and mRNA stability.
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Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus.
J. Microbiol.
PUBLISHED: 01-09-2013
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Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.
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Mucosal Vaccination with Recombinant Adenovirus Encoding Nucleoprotein Provides Potent Protection against Influenza Virus Infection.
PLoS ONE
PUBLISHED: 01-01-2013
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Influenza vaccines that target the highly variable surface glycoproteins hemagglutinin and neuraminidase cause inconvenience of having vaccination every year. For this reason, development of universal vaccines targeting conserved viral components is needed. In this study, we generated recombinant adenovirus (rAd) vaccine encoding nucleoprotein (NP) of A/PR/8/34 influenza virus, designated rAd/NP. BALB/c mice were immunized intranasally or sublingually with rAd/NP vaccine and subsequently challenged with lethal doses of heterologous as well as homologous influenza viruses. We found that intranasal immunization of rAd/NP elicited strong mucosal IgA responses as well as stronger CD8 T-cell responses toward immunodominant K(d)-restricted NP147-155 epitope than sublingual immunization. Importantly, only single intranasal but not sublingual immunization of rAd/NP provides potent protection against both homologous and heterologous influenza virus challenges. These results suggest that recombinant rAd/NP could be a universal vaccine candidate for mucosal administration against influenza virus.
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Screening novel biomarkers for metabolic syndrome by profiling human plasma N-glycans in Chinese Han and Croatian populations.
J. Proteome Res.
PUBLISHED: 10-18-2011
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N-glycans play an essential role in biological process and are associated with age, gender, and body mass parameters in Caucasian populations, whereas no study has been reported in Chinese populations. To investigate the correlation between N-glycan structures and metabolic syndrome (MetS) components, we conducted a population-based study in 212 Chinese Han individuals. The replication study was performed on 520 unrelated individuals from a Croatian island Kor?ula. The most prominent observation was the consistent positive correlations between different forms of triantennary glycans and negative correlations between glycans containing core-fucose with MetS components including BMI, SBP, DBP, and fasting plasma glucose (FPG) simultaneously. Significant differences in a number of N-glycan traits were also detected between normal and abnormal groups of BMI, BP, and FPG, respectively. In the multivariate analysis, the level of monosialylated glycans (structure loadings = -0.776) was the most correlated with the MetS related risk factors, especially with SBP (structure loadings = 0.907). Results presented here are showing that variations in the composition of the N-glycome in human plasma could represent the alternations of human metabolism and could be potential biomarkers of MetS.
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Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza.
PLoS ONE
PUBLISHED: 08-05-2011
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The ectodomain of matrix protein 2 (M2e) of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs) have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored.
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Rumen microbial response in production of CLA and methane to safflower oil in association with fish oil or/and fumarate.
Anim. Sci. J.
PUBLISHED: 04-15-2011
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Supplementation effect of fish oil and/or fumarate on production of conjugated linoleic acid (CLA) and methane by rumen microbes was examined when incubated with safflower oil. One hundred and twenty milligrams of safflower oil (SO), safflower oil with 24 mg fish oil (SOFO), safflower oil with 24 mmol/L fumarate (SOFA), or safflower oil with 24 mg fish oil and 24 mmol/L fumarate (SOFOFA) were added to the 90 mL culture solution. The culture solution was also made without any supplements (control). The SOFA and SOFOFA increased pH and propionate (C3) compared to other treatments from 3 h incubation time. An accumulated amount of total methane (CH(4) ) for 12 h incubation was decreased by all the supplements compared to control. The concentrations of c9,t11CLA for all the incubation times were increased in the treatments of SOFO, SOFA and SOFOFA compared to SO. The highest concentration of c9,t11CLA was observed from SOFOFA among all the treatments at all incubation times. Overall data indicate that supplementation of combined fumarate and/or fish oil when incubated with safflower oil could depress CH(4) generation and increase production of C(3) and CLA under the condition of current in vitro study.
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Characterization of Pt/a-plane GaN Schottky contacts using conductive atomic force microscopy.
J Nanosci Nanotechnol
PUBLISHED: 04-02-2011
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We investigated the local electrical properties of Pt Schottky contacts to a-plane n-type GaN using conductive atomic force microscopy (C-AFM). Current-voltage characteristics obtained by C-AFM showed rectifying properties, indicating nano-scale Schottky junction formation. Two-dimensional current maps revealed that the surface microstructures of GaN influenced transport properties of the junctions.
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Evaluation of protective efficacy of respiratory syncytial virus vaccine against A and B subgroup human isolates in Korea.
PLoS ONE
PUBLISHED: 03-12-2011
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Human respiratory syncytial virus (HRSV) is a significant cause of upper and lower respiratory tract illness mainly in infants and young children worldwide. HRSV is divided into two subgroups, HRSV-A and HRSV-B, based on sequence variation within the G gene. Despite its importance as a respiratory pathogen, there is currently no safe and effective vaccine for HRSV. In this study, we have detected and identified the HRSV by RT-PCR from nasopharyngeal aspirates of Korean pediatric patients. Interestingly, all HRSV-B isolates exhibited unique deletion of 6 nucleotides and duplication of 60 nucleotides in the G gene. We successfully amplified two isolates (KR/A/09-8 belonging to HRSV-A and KR/B/10-12 to HRSV-B) on large-scale, and evaluated the cross-protective efficacy of our recombinant adenovirus-based HRSV vaccine candidate, rAd/3xG, by challenging the immunized mice with these isolates. The single intranasal immunization with rAd/3xG protected the mice completely from KR/A/09-8 infection and partially from KR/B/10-12 infection. Our study contributes to the understanding of the genetic characteristics and distribution of subgroups in the seasonal HRSV epidemics in Korea and, for the first time, to the evaluation of the cross-protective efficacy of RSV vaccine against HRSV-A and -B field-isolates.
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Synthesis and biological evaluation of 4-piperidinecarboxylate and 4-piperidinecyanide derivatives for T-type calcium channel blockers.
Bioorg. Med. Chem. Lett.
PUBLISHED: 03-11-2011
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To obtain selective and potent inhibitor for T-type calcium channel by ligand based drug design, 4-piperidinecarboxylate and 4-piperidinecyanide derivatives were prepared and evaluated for in vitro and in vivo activity against ?(1G) calcium channel. Among them, several compounds showed good T-type calcium channel inhibitory activity and minimal off-target activity over hERG channel (% inhibition at 10 ?M=61.85-71.99, hERG channel IC(50)=1.57 ± 0.14-4.98 ± 0.36 ?M). Selected compound 31a was evaluated on SNL model of neuropathic pain and showed inhibitory effect on mechanical allodynia.
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Functional differences in pore properties between wild-type and cysteine-less forms of the CFTR chloride channel.
J. Membr. Biol.
PUBLISHED: 03-03-2011
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Studies of the structure and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel have been advanced by the development of functional channel variants in which all 18 endogenous cysteine residues have been mutated ("cys-less" CFTR). However, cys-less CFTR has a slightly higher single-channel conductance than wild-type CFTR, raising questions as to the suitability of cys-less as a model of the wild-type CFTR pore. We used site-directed mutagenesis and patch-clamp recording to investigate the origin of this conductance difference and to determine the extent of functional differences between wild-type and cys-less CFTR channel permeation properties. Our results suggest that the conductance difference is the result of a single substitution, of C343: the point mutant C343S has a conductance similar to cys-less, whereas the reverse mutation, S343C in a cys-less background, restores wild-type conductance levels. Other cysteine substitutions (C128S, C225S, C376S, C866S) were without effect. Substitution of other residues for C343 suggested that conductance is dependent on amino acid side chain volume at this position. A range of other functional pore properties, including interactions with channel blockers (Au[CN] (2) (-) , 5-nitro-2-[3-phenylpropylamino]benzoic acid, suramin) and anion permeability, were not significantly different between wild-type and cys-less CFTR. Our results suggest that functional differences between these two CFTR constructs are of limited scale and scope and result from a small change in side chain volume at position 343. These results therefore support the use of cys-less as a model of the CFTR pore region.
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Regulation of CFTR chloride channel macroscopic conductance by extracellular bicarbonate.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 10-06-2010
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The CFTR contributes to Cl? and HCO?? transport across epithelial cell apical membranes. The extracellular face of CFTR is exposed to varying concentrations of Cl? and HCO?? in epithelial tissues, and there is evidence that CFTR is sensitive to changes in extracellular anion concentrations. Here we present functional evidence that extracellular Cl? and HCO?? regulate anion conduction in open CFTR channels. Using cell-attached and inside-out patch-clamp recordings from constitutively active mutant E1371Q-CFTR channels, we show that voltage-dependent inhibition of CFTR currents in intact cells is significantly stronger when the extracellular solution contains HCO?? than when it contains Cl?. This difference appears to reflect differences in the ability of extracellular HCO?? and Cl? to interact with and repel intracellular blocking anions from the pore. Strong block by endogenous cytosolic anions leading to reduced CFTR channel currents in intact cells occurs at physiologically relevant HCO?? concentrations and membrane potentials and can result in up to ?50% inhibition of current amplitude. We propose that channel block by cytosolic anions is a previously unrecognized, physiologically relevant mechanism of channel regulation that confers on CFTR channels sensitivity to different anions in the extracellular fluid. We further suggest that this anion sensitivity represents a feedback mechanism by which CFTR-dependent anion secretion could be regulated by the composition of the secretions themselves. Implications for the mechanism and regulation of CFTR-dependent secretion in epithelial tissues are discussed.
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Effects of starch and protein sources on starch disappearance in the gastrointestinal tract of Hanwoo (Korean native) steers.
Anim. Sci. J.
PUBLISHED: 07-06-2010
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Hanwoo (Korean native) steers (274.8 +/- 4.6 kg) with ruminal and duodenal cannulae were used in a 4 x 4 Latin square design experiment to examine the effects of dietary treatments on starch disappearance in the gastrointestinal tract. Dietary treatments consisted of concentrate that were based on ground corn with soybean meal (C-SBM), ground corn with corn gluten meal (C-CGM), ground barley with soybean meal (B-SBM) and ground barley with corn gluten meal (B-CGM). Although the intakes of starch and protein for steers fed experimental diets were different, it did not change ruminal pH and total volatile fatty acid concentrations. Average duodenal CP flow and quantity of CP apparently digested post-ruminally was higher (P = 0.001) for CGM-based diets than SBM-based diets. There were increases in quantity (P < 0.001) and percentage (P < 0.001) of corn starch digested post-ruminally compared to barley starch. Synchronized diets showed higher percentages (P = 0.03) of starch apparently digested post-ruminally than asynchronization. Hanwoo steers fed a corn-based diet with a large quantity of starch reaching the duodenum and fed C-CGM supplying great amounts of protein to the small intestine may have contributed to increased post-ruminal starch digestion.
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Vertically oriented epitaxial germanium nanowires on silicon substrates using thin germanium buffer layers.
Nanotechnology
PUBLISHED: 06-29-2010
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We demonstrate a method to realize vertically oriented Ge nanowires on Si(111) substrates. Ge nanowires were grown by chemical vapor deposition using Au nanoparticles to seed nanowire growth via a vapor-liquid-solid growth mechanism. Rapid oxidation of Si during Au nanoparticle application inhibits the growth of vertically oriented Ge nanowires directly on Si. The present method employs thin Ge buffer layers grown at low temperature less than 600 degrees C to circumvent the oxidation problem. By using a thin Ge buffer layer with root-mean-square roughness of approximately 2 nm, the yield of vertically oriented Ge nanowires is as high as 96.3%. This yield is comparable to that of homoepitaxial Ge nanowires. Furthermore, branched Ge nanowires could be successfully grown on these vertically oriented Ge nanowires by a secondary seeding technique. Since the buffer layers are grown under moderate conditions without any high temperature processing steps, this method has a wide process window highly suitable for Si-based microelectronics.
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CdS/CdSe lateral heterostructure nanobelts by a two-step physical vapor transport method.
Nanotechnology
PUBLISHED: 03-10-2010
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The two-dimensional heterostructure nanobelts with a central CdSe region and lateral CdS structures are synthesized by a two-step physical vapor transport method. The large growth rate difference between lateral CdS structures on both +/- (0001) sides of the CdSe region is found. The growth anisotropy is discussed in terms of the polar nature of the side +/- (0001) surfaces of CdSe. High-resolution transmission electron microscopy reveals the CdSe central region covered with non-uniform CdS layer/islands. From micro-photoluminescence measurements, a systematic blueshift of emission energy from the central CdSe region in accordance with the increase of lateral CdS growth temperature is observed. This result indicates that the intermixing rate in the CdSe region with CdS increases with the increase of lateral CdS growth temperature. In conventional CdSSe ternary nanostructures, morphology and emission wavelength were correlated parameters. However, the morphology and emission wavelength are independently controllable in the CdS/CdSe lateral heterostructure nanobelts. This structure is attractive for applications in visible optoelectronic devices.
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Multiple mRNA decapping enzymes in mammalian cells.
Mol. Cell
PUBLISHED: 03-05-2010
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Regulation of RNA degradation plays an important role in the control of gene expression. One mechanism of eukaryotic mRNA decay proceeds through an initial deadenylation followed by 5 end decapping and exonucleolytic decay. Dcp2 is currently believed to be the only cytoplasmic decapping enzyme responsible for decapping of all mRNAs. Here we report that Dcp2 protein modestly contributes to bulk mRNA decay and surprisingly is not detectable in a subset of mouse and human tissues. Consistent with these findings, a hypomorphic knockout of Dcp2 had no adverse consequences in mice. In contrast, the previously reported Xenopus nucleolar decapping enzyme, Nudt16, is an ubiquitous cytoplasmic decapping enzyme in mammalian cells. Like Dcp2, Nudt16 also regulates the stability of a subset of mRNAs including a member of the motin family of proteins involved in angiogenesis, Angiomotin-like 2. These data demonstrate mammalian cells possess multiple mRNA decapping enzymes, including Nudt16 to regulate mRNA turnover.
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Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore.
J. Gen. Physiol.
PUBLISHED: 02-08-2010
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Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be "transplanted" to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl(-) conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby "rescuing" the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl(-) conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO(2))(4)(2-) in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl(-) channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl(-) transport through a combination of failure to increase Cl(-) conductance and increased susceptibility to channel block by cytosolic substances.
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Direct multiplex reverse transcription-nested PCR detection of influenza viruses without RNA purification.
J. Microbiol. Biotechnol.
PUBLISHED: 12-10-2009
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This paper describes the development a of direct multiplex reverse transcription-nested polymerase chain reaction (PCR) method, devised for simultaneous detection and typing of influenza viruses. This method combines the direct reverse transcription reaction without RNA purification with the enhancement of sensitivity and specificity of nested PCR. The method successfully detected three major human influenza viruses: influenza virus A subtype 1 (H1N1) and subtype 3 (H3N2), and influenza B virus (B). The minimum number of virus particles (pfu/ml) necessary for detection in spiked saliva samples was 200 (H1N1), 140 (H3N2), and 4.5 (B). The methods sensitivity and simplicity will be convenient for use in clinical laboratories for the detection and subtyping of influenza and possibly other RNA viruses.
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Proteomic analysis of endogenous conjugated linoleic acid biosynthesis in lactating rats and mouse mammary gland epithelia cells (HC11).
Biochim. Biophys. Acta
PUBLISHED: 04-14-2009
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This study was conducted to investigate the amount of CLA synthesized endogenously by rat mammary tissues in response to TVA (a precursor for cis-9, trans-11 CLA endogenous synthesis) treatment as well as the differences in the protein expression of genes encoding the biosynthesis of CLA in rat mammary tissue and mouse mammary gland epithelia cells (HC11). Treatment with TVA resulted in improved CLA productivity. Furthermore, 2-DE revealed two spots in samples of mammary tissues and one spot in samples of mammary gland epithelia cells (HC11) that were consistently altered in the TVA treatment groups when compared with the control group (non-fatty acid). The mRNA expression patterns of three of the proteins (PDI, PRDX2, LAMR1), as measured by real-time PCR, were similar to the pattern of protein abundance. In addition, the expression of SCD mRNA in the mammary tissue of rats and HC11 cell treated with TVA was higher than in the control group. Our results suggest that the identified proteins may be related to CLA biosynthesis in mammary tissue.
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Variations in conjugated linoleic acid (CLA) content of processed cheese by lactation time, feeding regimen, and ripening.
J. Agric. Food Chem.
PUBLISHED: 03-17-2009
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Dairy products are major sources of conjugated linoleic acid (CLA); thus, an increase in CLA content can improve the quality value of dairy products. The objective of this work was to determine the effects of lactation time, feeding regimen, and ripening period on the level of CLA in processed cheese. CLA content in milk varied with the period of lactation; high in spring (April and May, about 6.8 mg CLA/g fat) and relatively low in mid summer and winter (about 4.3 mg CLA/g fat). The effects of dietary regimen and ripening period were determined in milk, which was obtained from March to May. After aging for 4 months, the cheese made from milk obtained from cows fed on pasture contained relatively higher levels of CLA compared to cheese made from milk obtained from cows fed indoors (8.12 mg CLA/g fat vs 6.76 mg CLA/g fat), but there was no difference in 7 month-aged cheeses. In both pasture and indoor feeding, 7 month-aged cheeses showed higher CLA content than 4 month-aged cheeses. The contents of stearic acid (C18:0) and linolenic acid (C18:3) were significantly higher in cheese from pasture fed cows compared to those in cows fed indoors. These findings should be helpful for the efficient production of functional dairy products with high CLA contents.
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The growth and optical properties of CdSSe nanosheets.
Nanotechnology
PUBLISHED: 02-11-2009
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We have investigated the growth of ternary CdSSe nanostructures by physical vapor transport, specifically aiming at the synthesis of CdSSe nanosheets. CdSSe nanostructures with various S mole fractions are grown at growth temperatures less than 800 degrees C and photoluminescence from these nanostructures covers the entire visible spectral range. Morphological evolution from nanowires to nanosheets is observed as increasing growth temperatures due to the change of relative dominance in the growth mechanism from vapor-liquid-solid to vapor-solid. Nanosheets whose widths extend several tens of micrometers are successfully synthesized. However, nanosheets show a strong suppression effect of S incorporation when the growth is dominated by the vapor-solid mechanism. A micro-photoluminescence study shows high optical quality of individual CdSSe nanostructures with polarization anisotropy ratios rho ~ 0.1. In addition, lateral heterostructure CdS/CdSe nanosheets are successfully synthesized by two step growth. Strong red and green emissions are observed by selective laser excitations focusing at the center and edge parts of these heterostructure nanosheets, respectively. These heterostructure CdS/CdSe nanosheets may be useful for multicolor light emitting devices.
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Intranasal delivery of cholera toxin induces th17-dominated T-cell response to bystander antigens.
PLoS ONE
PUBLISHED: 01-14-2009
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Cholera toxin (CT) is a potent vaccine adjuvant, which promotes mucosal immunity to protein antigen given by nasal route. It has been suggested that CT promotes T helper type 2 (Th2) response and suppresses Th1 response. We here report the induction of Th17-dominated responses in mice by intranasal delivery of CT. This dramatic Th17-driving effect of CT, which was dependent on the B subunit, was observed even in Th1 or Th2-favored conditions of respiratory virus infection. These dominating Th17 responses resulted in the significant neutrophil accumulation in the lungs of mice given CT. Both in vitro and in vivo treatment of CT induced strongly augmented IL-6 production, and Th17-driving ability of CT was completely abolished in IL-6 knockout mice, indicating a role of this cytokine in the Th17-dominated T-cell responses by CT. These data demonstrate a novel Th17-driving activity of CT, and help understand the mechanisms of CT adjuvanticity to demarcate T helper responses.
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Induction of BAFF expression by IFN-? via JAK/STAT signaling pathways in human intestinal epithelial cells.
J. Leukoc. Biol.
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BAFF plays an important role in the development of B cells. Here, we investigated the effect of IFN-? on BAFF expression in human intestinal epithelial cells. IFN-? induced soluble and membrane-bound BAFF production in a dose- and time-dependent manner. IFN-?-induced BAFF release from polarized intestinal epithelial cells was observed in apical and basolateral compartments. JAK I inhibitor suppressed IFN-?-induced BAFF expression. Moreover, IFN-? enhanced STAT1 phosphorylation and expression of IRF-1. Transient transfection and reporter gene assay showed that the BAFF promoter region spanning -750 to -500 bp from the translation initiation site was crucial for IFN-?-induced BAFF expression. Nucleotide sequence analysis revealed a GAS element in the promoter region. ChIP assay confirmed the enhanced binding of phosphorylated STAT1 to the BAFF promoter region at -800 to -601 bp. Furthermore, IFN-? enhanced DNA binding to GAS and its transcriptional activation, as determined by the EMSA and reporter gene assay. Collectively, these results suggest that IFN-? induces BAFF expression in human intestinal epithelial cells through JAK/STAT signaling pathways that might activate the GAS and IRF-1-binding element in the BAFF promoter.
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5-Phenyl-1,3,4-oxadiazol-2-amine.
Acta Crystallogr Sect E Struct Rep Online
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In the title complex, C(8)H(7)N(3)O, the C-O [1.369?(2) and 1.364?(3)?Å] and C=N [1.285?(3) and 1.289?(3)?Å] bond lengths in the oxadiazole ring are each almost identical within systematic errors, although different substituents are attached to the ring. The phenyl ring is inclined to the planar oxadiazole ring [r.m.s. deviation 0.002?Å] by 13.42?(18)°. In the crystal, molecules are linked via N-H?N hydrogen bonds, forming double-stranded chains propagating along [010].
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Sublingual immunization with recombinant adenovirus encoding SARS-CoV spike protein induces systemic and mucosal immunity without redirection of the virus to the brain.
Virol. J.
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Sublingual (s.l.) administration of soluble protein antigens, inactivated viruses, or virus-like particles has been shown to induce broad immune responses in mucosal and extra-mucosal tissues. Recombinant replication-defective adenovirus vectors (rADVs) infect mucosa surface and therefore can serve as a mucosal antigen delivery vehicle. In this study we examined whether s.l. immunization with rADV encoding spike protein (S) (rADV-S) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) induces protective immunity against SARS-CoV and could serve as a safe mucosal route for delivery of rADV.
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Efficiency enhancement in a-plane InGaN/GaN light emitters with carbon nanotubes.
J Nanosci Nanotechnol
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This study investigates the coupling modes of a-plane InGaN/GaN mutiquantum wells (MQWs) with single-walled carbon nanotubes (SWCNTs). The enhancement of light emissions at resonance photon energies can be explained by the surface plasmon coupling of the MQW-SWCNT hybrid structure. The photoluminescence (PL) enhancement ratios of the indigo (2.90 eV) emission from MQWs with SWCNTs reveal three coupling modes at 2.50 eV, 2.97 eV, and 3.42 eV. In addition, the trend of the PL intensity ratios and efficiencies corresponds to that of the PL enhancement ratios. The PL efficiencies for the green (2.46 eV) and indigo (2.90 eV) emissions of SWCNT-coated MQWs are 32% and 110% better than the corresponding values of uncoated MQWs, respectively. The results show that the MQW-SWCNT hybrid structure has the potential to be applied in high-efficiency light emitters in the visible and ultraviolet range.
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[Mitochondria couple cellular Ca(2+) signal transduction].
Sheng Li Xue Bao
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It has been shown that mitochondria not only control their own Ca(2+) concentration ([Ca(2+)]), but also exert an influence over Ca(2+) signaling of the entire cell, including the endoplasmic reticulum or the sarcoplasmic reticulum, the plasma membrane, and the nucleus. That is to say, mitochondria couple cellular metabolic state with Ca(2+) transport processes. This review focuses on the ways in which the mitochondrial Ca(2+) handling system provides integrity and modulation for the cell to cope with the complex actions throughout its life cycle, enumerates some indeterminate aspects about it, and finally, prospects directions of future research.
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Effect of maternal immune status on responsiveness of bacillus calmette-gurin vaccination in mouse neonates.
Osong Public Health Res Perspect
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Bacillus Calmette-Guérin (BCG) vaccination has proven to be efficient in immunologically naïve infants; however, it has not been investigated that maternal natural exposure to Mycobacterium and/or BCG vaccine could influence the characteristics of immune responses to BCG in newborns. In this study, we analyzed whether the maternal immune status to M tuberculosis (M tb) can affect neonatal immunity to BCG using a mouse model.
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Pseudohalide anions reveal a novel extracellular site for potentiators to increase CFTR function.
Br. J. Pharmacol.
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There is great interest in the development of potentiator drugs to increase the activity of the cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis. We tested the ability of several anions to potentiate CFTR activity by a novel mechanism.
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The effect of aging and severity of sleep apnea on heart rate variability indices in obstructive sleep apnea syndrome.
Psychiatry Investig
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This study aims to analyze how much heart rate variability (HRV) indices discriminatively respond to age and severity of sleep apnea in the obstructive sleep apnea syndrome (OSAS).
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Dual role of respiratory syncytial virus glycoprotein fragment as a mucosal immunogen and chemotactic adjuvant.
PLoS ONE
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Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.
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