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Bacteriophage T7 encodes its own DNA polymerase, the product of gene 5 (gp5). In isolation, gp5 is a DNA polymerase of low processivity. However, gp5 becomes highly processive upon formation of a complex with Escherichia coli thioredoxin, the product of the trxA gene. Expression of a gp5 variant in which aspartate residues in the metal-binding site of the polymerase domain were replaced by alanine is highly toxic to E. coli cells. This toxicity depends on the presence of a functional E. coli trxA allele and T7 RNA polymerase-driven expression but is independent of the exonuclease activity of gp5. In vitro, the purified gp5 variant is devoid of any detectable polymerase activity and inhibited DNA synthesis by the replisomes of E. coli and T7 in the presence of thioredoxin by forming a stable complex with DNA that prevents replication. On the other hand, the highly homologous Klenow fragment of DNA polymerase I containing an engineered gp5 thioredoxin-binding domain did not exhibit toxicity. We conclude that gp5 alleles encoding inactive polymerases, in combination with thioredoxin, could be useful as a shutoff mechanism in the design of a bacterial cell-growth system.

The foundational adenine base editors (for example, ABE7.10) enable programmable A•T to G•C point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG protospacer adjacent motif (PAM) sites, ABE8s result in ~1.5× higher editing at protospacer positions A5-A7 and ~3.2× higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2 with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification, which is maintained when multiplexed across three loci. Delivered as messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

This report lays the foundation stone for the real time detector to control and monitor antibiotic resistant bacteria’s. To start with, firstly, the green synthesis of silver nanoparticles were done by the bio-reduction of silver nitrate using different concentrations of medicinal plant extract taken from the leaves of Azadirachta indica (neem). The well-known standard surface plasmon resonance (SPR) wavelength of 430 nm and the characteristic brown color of the solution confirms the formation of eco-friendly silver nanoparticles. The silver nanoparticle size in the range of 10–20 nm was observed from TEM measurements for the sample showing best SPR which was further selected for conjugation with QDs. Secondly glutathione capped ZnS:Mn QDs were synthesized and conjugated with silver nanoparticles (0.09–0.1 mg/mL) and then their zone of inhibition studies were done using antibiotic resistant gram positive as well as gram negative bacteria’s i.e. Staphylococcus aureus and Escherichia coli respectively. Lastly, time resolved photoluminescence studies of the conjugated system were done to correlate zone of inhibition (antibiotic sensitivity) with silver nanoparticle concentration in both the cases of a gram positive bacteria and gram negative bacteria. Luminescence intensity of glutathione capped QDs conjugated silver nanoparticles decreases with increasing concentration of silver nanoparticles in tune with the increasing zone of inhibition (antibiotic sensitivity). This is very important for making a fluorescence based detector to monitor antibiotic resistant bacteria’s.

The Helicobacter pylori type IV secretion system (T4SS) encoded on the cag pathogenicity island (cagPAI) secretes the CagA oncoprotein and other effectors into the gastric epithelium. During murine infection, T4SS function is lost in an immune-dependent manner, typically as a result of in-frame recombination in the middle repeat region of cagY, though single nucleotide polymorphisms (SNPs) in cagY or in other essential genes may also occur. Loss of T4SS function also occurs in gerbils, nonhuman primates, and humans, suggesting that it is biologically relevant and not simply an artifact of the murine model. Here, we sought to identify physiologically relevant conditions under which T4SS function is maintained in the murine model. We found that loss of H. pylori T4SS function in mice was blunted by systemic Salmonella coinfection and completely eliminated by dietary iron restriction. Both have epidemiologic parallels in humans, since H. pylori strains from individuals in developing countries, where iron deficiency and systemic infections are common, are also more often cagPAI+ than strains from developed countries. These results have implications for our fundamental understanding of the cagPAI and also provide experimental tools that permit the study of T4SS function in the murine model. IMPORTANCE The type IV secretion system (T4SS) is the major Helicobacter pylori virulence factor, though its function is lost during murine infection. Loss of function also occurs in gerbils and in humans, suggesting that it is biologically relevant, but the conditions under which T4SS regulation occurs are unknown. Here, we found that systemic coinfection with Salmonella and iron deprivation each promote retention of T4SS function. These results improve our understanding of the cag pathogenicity island (cagPAI) and provide experimental tools that permit the study of T4SS function in the murine model.

Volatile compositions and sensory characteristics of 11 commercially distilled soju samples were investigated using headspace solid-phase microextraction (HS-SPME) with gas chromatography-mass spectrometry (GC-MS) and sensory descriptive analysis. A total of 59 major volatile compounds, consisting of 32 esters, 10 alcohols, 2 acids, 5 aldehydes, 3 ketones, 1 hydrocarbon, 1 furan, 2 phenols, and 3 miscellaneous compounds, were identified. From the principal component analysis (PCA) of volatile data, MSJ made by atmospheric distillation showed a clear distinction in volatile compositions compared to that of other samples made by vacuum distillation. Based on PCA of the sensory data determined by a panel of ten judges, MSJ was associated with a large amount of longer chain esters that showed high intensities in bitter taste and yeast/nuruk-related flavor attributes. HYJ, LPJ, and HAJ made with rice as a raw material were associated with lower intensities of the alcohol aroma, while JRJ and OKJ aged in oak barrels were associated with fruit flavor, sweet flavor, and brandy aroma. In the partial least squares regression (PLSR) analysis to see any relationship between volatile and sensory data, longer chain esters like ethyl tetradecanoate, and ethyl hexadecanoate were highly associated with bleach aroma. In contrast, positive correlations were seen with barley aroma and yeast flavor with hexanal, nonanal, benzaldehyde, and 2-methoxy-phenol.

This study analyzed the dietary pattern of Southeast Asian workers (Vietnamese, Thais, Cambodians and Myanmar) living in South Korea in order to recognize the dietary changes after they moved to South Korea. Questionnaires were completed by 251 Southeast Asian workers living in South Korea. Using a self-administered questionnaire, we assessed the diets before and after living in the hometown and in South Korea. Significant changes observed in the Southeast Asian workers were decreased in consumption frequency of fresh fruits, cooked vegetables, rice noodles, green tea and glutinous rice, and increase in consumption of Kimchi, seaweed, milk, coffee and pizza. These changes were attributed to rapid dietary acculturation. The frequencies of eating homemade food were significantly decreased after they came to Korea except for Thais. Thais showed the highest frequencies of eating homemade food daily among others. 28.2% of respondents said their health condition had deteriorated after living in South Korea due to difficulties to adapt Korean food, increased frequencies of eating instant food, and lacking exercises. By providing understanding of the dietary patterns of Southeast Asian workers, these results can be used for preliminary data to develop a program for their Korean food adaptation.

The development of a subunit Salmonella vaccine has been hindered by the absence of detailed information about antigenic targets of protective Salmonella-specific T and B cells. Recent studies have identified SseB as a modestly protective Ag in susceptible C57BL/6 mice, but the mechanism of protective immunity remains undefined. In this article, we report that simply combining Salmonella SseB with flagellin substantially enhances protective immunity, allowing immunized C57BL/6 mice to survive for up to 30 d following challenge with virulent bacteria. Surprisingly, the enhancing effect of flagellin did not require flagellin Ag targeting during secondary responses or recognition of flagellin by TLR5. Although coimmunization with flagellin did not affect SseB-specific Ab responses, it modestly boosted CD4 responses. In addition, protective immunity was effectively transferred in circulation to parabionts of immunized mice, demonstrating that tissue-resident memory is not required for vaccine-induced protection. Finally, protective immunity required host expression of IFN-γR but was independent of induced NO synthase expression. Taken together, these data indicate that Salmonella flagellin has unique adjuvant properties that improve SseB-mediated protective immunity provided by circulating memory.

For the development of elderly-friendly food, the physicochemical properties and preferences of cooked kkakdugi prepared with various blanching times were examined. As fermentation progressed, significant differences in samples were observed with overall decreasing trends of pH, reducing sugar, lightness (L), yellowness (b), and hardness. Titratable acidity, redness (a), and lactic acid increased during the storage time. The preference test showed that the sample blanched for 3 min (SK3) scored the highest in aroma, taste, texture, and overall acceptance. Overall, SK3 was the most preferred as cooked kkakdugi, which helps elderly people suffering from mastication and deglutition.

We investigated the effects of Sarcodon aspratus, Agaricus bisporus, and Lentinula edodes aqueous extracts on the tenderization of bovine longissimus dorsi muscle. Meat quality and muscle protein degradation were examined as well. Beef chunks were marinated in distilled water (control), 5% S. aspratus (SA), 5% A. bisporus (AB), or 5% L. edodes (LE) extracts. SA was shown to have a higher enzymatic activity (p

Nucleotide excision repair (NER) is an essential DNA repair system distinguished from other such systems by its extraordinary versatility. NER removes a wide variety of structurally dissimilar lesions having only their bulkiness in common. NER can also repair several less bulky nucleobase lesions, such as 8-oxoguanine. Thus, how a single DNA repair system distinguishes such a diverse array of structurally divergent lesions from undamaged DNA has been one of the great unsolved mysteries in the field of genome maintenance. Here we employ a synthetic crystallography approach to obtain crystal structures of the pivotal NER enzyme UvrB in complex with duplex DNA, trapped at the stage of lesion-recognition. These structures coupled with biochemical studies suggest that UvrB integrates the ATPase-dependent helicase/translocase and lesion-recognition activities. Our work also conclusively establishes the identity of the lesion-containing strand and provides a compelling insight to how UvrB recognizes a diverse array of DNA lesions.

Background The use of lactobacillus probiotics has been proposed as an alternative to prophylactic antibiotics for preventing urinary tract infection (UTI) in the era of antibiotic resistance. In this study, the antimicrobial activity of lactobacillus strains against uropathogens, was evaluated and compared with that of antibiotics. Methods To evaluate inhibitory activities of lactobacilli against uropathogens, six lactobacillus strains (L. gasseri, L. rhamnosus, L. acidophilus, L. plantarum, L. paracasei, L. acidophilus) and four representative uropathogens of infantile UTI (extended-spectrum beta-lactamase [ESBL](−) Escherichia coli, ESBL(+) E. coli, Proteus vulgaris, Enterococcus fecalis) were selected. Lactobacillus strain in vitro inhibition of each uropathogen was evaluated on MRS agar well diffusion assay and compared with that of commercial antibiotic discs. Results Average inhibitory zone for each of the six lactobacillus strains against the four uropathogens showed slightly different but consistent inhibition (inhibitory zone diameter, 10.5–20.0 mm). This was different to that of the antibiotic discs, which had a wider range of inhibition (inhibitory zone diameter

Volatile compounds of 7 commercial fermentation starters, including 4 traditional nuruk, 2 ipguk, and 1 crude amylolytic enzyme, were extracted using solvent-assisted flavor evaporation (SAFE), and extracts were analyzed using GC-MS. A total of 70 volatile compounds, including 4 esters, 10 acids, 16 alcohols, 16 hydrocarbons, 9 ketones, 5 aldehydes, 3 volatiles phenols, and 7 miscellaneous components were identified. Concentrations of volatile compounds, including 4,6-dimethylundecane, methyl 2-hydroxybenzoate, 1-hexanol, 2-phenylethanol, 3-methylbutanoic acid, and hexanoic acid were higher in 4 traditional nuruk starters than in others. The major volatile components in 2 ipguk starters were the hydrocarbones dodecane, heptadecane, octadecane, and nonadecane. Concentrations of 2-methoxy-4-vinylphenol and 3-hydroxy-2-butanone were highest in the crude amylolytic enzyme starter. Compositions and amounts of volatile compounds were different among the 7 commercial fermentation starters.

The formation of a replication loop on the lagging strand facilitates coordinated synthesis of the leading- and lagging-DNA strands and provides a mechanism for recycling of the lagging-strand DNA polymerase. As an Okazaki fragment is completed, the loop is released, and a new loop is formed as the synthesis of a new Okazaki fragment is initiated. Loop release requires the dissociation of the complex formed by the interactions among helicase, DNA polymerase, and DNA. The completion of the Okazaki fragment may result in either a nick or a single-stranded DNA region. In the replication system of bacteriophage T7, the dissociation of the polymerase from either DNA region is faster than that observed for the dissociation of the helicase from DNA polymerase, implying that the replication loop is released more likely through the dissociation of the lagging-strand DNA from polymerase, retaining the polymerase at replication fork. Both dissociation of DNA polymerase from DNA and that of helicase from a DNA polymerase · DNA complex are much faster at a nick DNA region than the release from a ssDNA region. These results suggest that the replication loop is released as a result of the nick formed when the lagging-strand DNA polymerase encounters the previously synthesized Okazaki fragment, releasing lagging-strand DNA and retaining DNA polymerase at the replication fork for the synthesis of next Okazaki fragment.

Activating mutations in RAS proteins occur in ~1/3 of human cancers. These mutations impair the ability of the protein to hydrolyze GTP to GDP. As a result, mutant RAS proteins exist predominantly in the GTP-bound state, which directly activates aberrant downstream signaling via interaction with effectors such as RAF. Most RAS mutations occur at glycine 12 of the KRAS isoform. One such mutation, KRAS G12C, is particularly common in non-small cell lung cancer where it is found in ~15% of lung adenocarcinomas. Recent efforts have targeted KRAS G12C in the GDP-bound state; however, direct pharmacologic inhibition of active, GTP-bound KRAS G12C has proved challenging. Here, we deployed a novel SMARTTM (Small Molecule Assisted Receptor Targeting) platform to advance covalent compounds that selectively inhibit GTP-bound KRAS G12C. Using a mechanism reminiscent of the natural products rapamycin and cyclosporine, these compounds promote formation of a novel inhibitory ternary complex consisting of cyclophilin A (CypA, an abundant immunophilin present in all human cells), the SMART inhibitor, and GTP-KRAS G12C. Structure-based design of the SMART inhibitor yielded potent covalent inhibitors of GTP-KRAS G12C that exhibit >100-fold selectivity for mutant KRAS G12C over WT KRAS. Structural analysis of the ternary complex revealed that the covalent linkage between the SMART inhibitor and the mutant cysteine of KRAS occurred in the context of extensive interactions between CypA, the SMART inhibitor, and GTP-KRAS G12C that provide significant binding affinity (KI = 2.5 μM). The GTP-KRAS G12C|Inhibitor|CypA complex directly occluded effector binding, and as such, the compounds disrupted the KRAS-RAF interaction in biochemical assays. This activity was dependent on CypA, underlining the importance of the KRAS G12C| CypA protein-protein interaction in driving target engagement. In cell-based studies, SMART inhibitors crosslinked KRAS G12C and potently inhibited ERK phosphorylation and cell growth in G12C mutant tumor cell lines but had no effect on non-G12C bearing tumor cells. CRISPR knockout of cellular CypA confirmed that these activities were dependent on the presence of endogenous CypA. Importantly, SMART inhibitors bind directly to active, GTP-KRAS G12C and thus, their activity does not rely on trapping KRAS G12C in the inactive GDP-bound state. As a result, the cellular potency of SMART inhibitors with respect to crosslinking, pERK inhibition, and growth inhibition was maintained in the presence of growth factor treatments that reduce the cellular GDP-KRAS G12C pool. In contrast, we found that the activity of a previously described GDP-KRAS G12C targeting inhibitor was attenuated by growth factor treatment. To our knowledge, these are the first examples of mutant-selective KRAS inhibitors that target the active, GTP-bound state of KRAS G12C. We are currently optimizing the drug-like properties of these SMART inhibitors and evaluating their activity in in vivo models. Citation Format: Michelle L. Stewart, Nicholas R. Perl, Seung-Joo Lee, Linlong Xue, Minyun Zhou, Jonah Simon, Kathryn M. Luly, Siminia Grigoriu, Alex Yuzhakov, Alec Silver, Jason T. Lowe, Cindy C. Benod, Alan S. Mann, Gregory L. Verdine, Alan C. Rigby, Mark J. Mulvihill, Earl W. May, Anna Kohlmann, Sharon A. Townson, Roy M. Pollock, Meizhong Jin. Development of inhibitors of the activated form of KRAS G12C [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B37.

Bacteriophage T7 uses the thioredoxin of its host, Escherichia coli, to enhance the processivity of its DNA polymerase, a requirement for the growth of phage T7. The evolutionarily conserved structure and high degree of homology of amino acid sequence of the thioredoxin family imply that homologues from other organisms might also interact with T7 DNA polymerase to support the phage growth. Despite the structural resemblance, human thioredoxin, whose X-ray crystallographic structure overlaps with that of the E. coli protein, cannot support T7 phage growth. It does not form a complex with T7 DNA polymerase as determined by surface plasmon resonance and thus does not increase the processivity. Homologous scanning analysis using this nonfunctional homologue reveals that the 60 N-terminal and the 12 C-terminal amino acid residues of E. coli thioredoxin can be substituted for its human counterpart without significantly affecting phage growth. Comparison of chimeric thioredoxins, followed by site-directed mutagenesis, identifies leucine 95 as a critical element. This residue may contribute to hydrophobic interactions with the thioredoxin-binding loop of the polymerase; levels of DNA binding and thus nucleotide polymerization are significantly decreased in the absence of this residue. The results suggest that the specific interactions at the interface of thioredoxin and DNA polymerase, rather than the overall structure, are important in the interactions that promote high processivity.

The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site.

Most pathogens infect through mucosal surfaces, and parenteral immunization typically fails to induce effective immune responses at these sites. Development of oral-administered vaccines capable of inducing mucosal as well as systemic immunity while bypassing the issues of antigen degradation and immune tolerance could be crucial for the control of enteropathogens. This study demonstrates that U-Omp19, a bacterial protease inhibitor with immunostimulatory features, coadministered with Salmonella antigens by the oral route, enhances mucosal and systemic immune responses in mice. U-Omp19 was able to increase antigen-specific production of IFN-γ and IL-17 and mucosal (IgA) antibody response. Finally, oral vaccination with U-Omp19 plus Salmonella antigens conferred protection against virulent challenge with Salmonella Typhimurium, with a significant reduction in bacterial loads. These findings prove the efficacy of this novel adjuvant in the Salmonella infection model and support the potential of U-Omp19 as a suitable adjuvant in oral vaccine formulations against mucosal pathogens requiring T helper (Th)1-Th17 protective immune responses. Fil: Risso, Gabriela Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Carabajal, Marianela Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Bruno, Laura Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Ibañez, Andres Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Coria, Mirta Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Pasquevich, Karina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Lee, Seung Joo. University of California at Davis; Estados Unidos Fil: McSorley, Stephen J.. University of California at Davis; Estados Unidos Fil: Briones, Carlos Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina Fil: Cassataro, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentina

Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR-signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin-specific CD4(+) T-cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin-specific T-cell responses.

Seung-Joo Lee*Department of Culinary and Food Service Management, Sejong UniversityAbstract In this study, 100 consumers (men, 50; women, 50; age group, 20-50 years) rated their overall preferences for 24Korean raspberry wines by using a 9-point hedonic scale. The analysis of variance was constructed to evaluate the effect ofgender, age, and samples on the preference scores of the wine products. Significant differences were observed in overallpreferences for the 24 samples; however, no interactions based on preferences by age and gender groups were noted. Clusteranalysis was performed to determine sample clustering based on the frequencies from the preference data. Three clusters wereobtained; these three clusters were well separated based on the mean overall preference scores for the samples. Discriminantanalysis based on the three clusters also confirmed the same grouping of samples with 100% accuracy.Keywords: Korean black raspberry wine, preference, classification, discriminant analysis, quality

T cell effector functions can be elicited by non-cognate stimuli, but the mechanism and contribution of this pathway to the resolution of intracellular-macrophage infections has not been defined. Here we have shown that CD4+ T helper-1 (Th1) cells can be rapidly stimulated by microbe-associated molecular patterns (MAMPs) during active infection with Salmonella or Chlamydia. Further, maximal stimulation of Th1 cells by lipopolysaccharide (LPS) did not require T cell-intrinsic expression of toll-like receptor-4 (TLR4), interleukin-1 receptor (IL-1R), or interferon-γ receptor (IFN–γR), but instead required the adaptor protein Myd88, IL-18R, and IL-33R. Innate stimulation of Th1 cells also required host expression of TLR4 and inflammasome components that together increased serum concentrations of IL-18. Finally, the elimination of non-cognate Th1 cell stimulation hindered the resolution of primary Salmonella infection. Thus, the in vivo bactericidal capacity of Th1 cells is regulated by the response to non-cognate stimuli elicited by multiple innate immune receptors.

The volatile compounds and physicochemical properties of rice beers brewed with three medicinal plants, namely Acanthopanax senticosus (Siberian ginseng), Scutellaria baicalensis (baikal skullcap) and Cornus officinalis (Japanese cornel) were analysed. The rice beers were produced and fermented from unhulled ground rice, malt, and medicinal plant extracts. The medicinal plant extracts, used at 5 and 10 % (v/v) were blended with wort before fermentation. Compared with a draft beer fermented without rice, the rice beers had lower levels of pH, acidity, amino acid content and reducing sugars. Of the rice beers, the beer with the addition of 10% Japanese cornel had the highest colour value and foam stability. The volatile compounds of the rice beers were extracted using a solvent-assisted flavour evaporation apparatus and analysed by gas chromatography–mass spectrometry. The rice beer with 10% Siberian ginseng showed the most diverse volatile profile, in that 54 kinds of volatile compounds were detected. The rice beer with 10% Japanese cornel had the highest relative amount of volatile chemicals of all of the rice beers. Lactones such as γ-hexalactone, γ-nonalactone and γ-decalactone were detected in all of the rice beers. Copyright © 2013 The Institute of Brewing & Distilling

Seung-Joo Lee*Department of Culinary and Food Service Management, Sejong UniversityAbstract Korean black raspberry fruits (bokbunja) were collected from 11 different producing regions, and commercialblack raspberry wines were obtained from 24 manufacturers. Samples were analyzed for soluble solids, titratable acidity,pH, reducing sugar content, hunter color values, intensity, hue, total phenolic content, and organic acids. The fruit samplesshowed similar pH levels (3.43-3.52), but significantly differed in total acidity levels (9.98-16.2 g/L). The predominantorganic acids in the fruit samples were citric acids (1.39-5.63 mg/mL) and succinic acids (0.25-6.53 mg/mL). Among thesamples, black raspberry fruits from Goksung and Nonsan showed the lowest levels of total phenolic content, and thelowest values in intensity. The fruits from Jeongeup and Sunchang showed higher levels of phenolic content, soluble solids,and intensity. Some wine samples, including BH, KO, SR, and SE, showed overall high levels of phenolic content, organicacids like citric and succinic acid, and color values such as a*, b*, and intensity. Other wine samples, including DW, SC,GJ, and NB, were high in acetic acid, color values like L*, and hue.Keywords: Korean black raspberry, bokbunja, Korean black raspberry wine, physico-chemical composition, regionality

Objective : Allergic rhinitis(AR) is an inflammation or irritaion of nasal mucosa. Usually, serum cytokine is regarded as one of the most important factors in AR. Coisis semen have been used for the diseases in digestive organs in traditional chinese medicine. Nowaday, there were some studies reported about the effects of coisis semens on serum cytokine. However the results of theses studies were varies, and moreover there was no study which have used AR animal model. Therefore this study is aimed to determine therapeutic effects of coisis semen extract by observing changes of serum cytokine(IgE, TNF-, IL-, IFN- and IL-13) with AR animal model. Methods : Thirty BALB/c mice were divided into six groups : Normal group, Control group, sample I, sample II, sample III, sample IV. Except for Normal group, all the mice in other 5 groups were sensitized intraperionealy by 0.1% ovalbumin solution three times at intervals of one week. Then intranasal sensitization was performed by diffusing 0.1% ovalbumin solution three times at intervals of two days. Normal group were used Normal Saline instead of ovalbumin solution. After the first day of study, sample I, sample II, sample III, and sample IV were orally administrated coisis semen extract by 100mg/kg, 200mg/kg, 500mg/kg, and 1g/kg respectively for 28days. Then, the changes of serum IgE, TNF-, IL-, IFN- and IL-13 were observed in 6 groups. We used the statistical methods of ANOVA, post hoc by duncun, and Kruskal-Wallis test(p. But, there were no statistical changes in IL-, IL-13 and IFN-. Conclusions : According to above results, it is supposed that coisis semen extract has not some significant effects on cytokine of AR animal model. There was no evidence for using coisis semen in relieving symptoms of allergic rhinitis.

SummaryThe Wnt/β-catenin signaling pathway plays a major role in tissue homeostasis, and its dysregulation can lead to various human diseases. Aberrant activation of β-catenin is oncogenic and is a critical driver in the development and progression of human cancers. Despite the significant potential of targeting the oncogenic β-catenin pathway for cancer therapy, the development of specific inhibitors remains insufficient. Using a T cell factor (TCF)-dependent luciferase-reporter system, we screened for small-molecule compounds that act against Wnt/β-catenin signaling and identified MSAB (methyl 3-{[(4-methylphenyl)sulfonyl]amino}benzoate) as a selective inhibitor of Wnt/β-catenin signaling. MSAB shows potent anti-tumor effects selectively on Wnt-dependent cancer cells in vitro and in mouse cancer models. MSAB binds to β-catenin, promoting its degradation, and specifically downregulates Wnt/β-catenin target genes. Our findings might represent an effective therapeutic strategy for cancers addicted to the Wnt/β-catenin signaling pathway.

Protective immunity against Salmonella infection is known to require CD4 Th1 cells and B cells, but the role of MHC class-I-restricted CD8 T cells is less clear. Previous studies have suggested that CD8 T cells participate in secondary, but not primary, bacterial clearance. However, these studies have used experimental models that are difficult to interpret and do not clearly isolate the role of MHC class-I-restricted CD8 T cells from other cell populations. Here, we examined the role of class-I-restricted T cells in protection against Salmonella infection using mice lacking all classical MHC class-Ia molecules, perforin, or granzyme B. Immunized K b D b -, perforin-, granzyme B-, or perforin/granzyme B-deficient mice were able to resolve secondary infection with virulent Salmonella , demonstrating that class-I-restricted CTLs are not required for acquired immunity. However, during primary infection with attenuated bacteria, bacterial clearance was delayed in each of these mouse strains when compared to wild-type mice. Taken together, these data demonstrate that CD8 T cells are not required for acquired immunity to Salmonella , but can play a protective role in resolving primary infection with attenuated bacteria.

The zinc-binding domain (ZBD) of prokaryotic DNA primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA primase to catalyze template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis.

+ and NO3 - -N ranges than that of the control, and the average of the lightly thinned site was significantly higher than that of the control for the other two thinning treatment. Average NH4 + concentrations were higher than the average NO3 - concentrations in all study sites, and the of extractable NH4 + concentrations were higher in the growing season of Jun and July, and then declined in the late summer and fall seasons. Nitrification rates were comparatively low and net N minerali- zation differed significantly by thinning management, and showed seasonal variations. For net N minerali- zation and nitrification rate during the study period, the lightly thinned site had a significantly higher aver- age than the other three sites.

An efficient and rapid colorimetric method was established for the selection of tea tree lines rich in polyphenol from 160 tea tree lines. The efficient selection method was made possible by the adoption of the Folin-Ciocalteu (FC) colorimetric method for qualitative detection of the polyphenols. The polyphenol composition varied widely among the randomly sampled 160 tea tree lines. The tea tree lines were classified into three groups on the basis of color intensity obtained after treatment of tea extracts with FC reagent. The polyphenol content of the three groups was quantified and validated by UV spectrophotometric analysis. Among various tea lines, H-23 tea line exhibited the highest amounts of polyphenol that was 2.89-fold higher than those found in low polyphenol producing tea tree (H-9). Hence FC colorimetric method can become a reliable selection method for easy and rapid selection of tea population rich in polyphenol in a tea tree breeding programme.

Recent studies have shed light on the connection between elevated erythropoetin production/spleen erythropoiesis and increased susceptibility to Salmonella infection. In this article, we provide another mouse model, the SIRPα-deficient (Sirpα−/−) mouse, that manifests increased erythropoiesis as well as heightened susceptibility to Salmonella infection. Sirpα−/− mice succumbed to systemic infection with attenuated Salmonella, possessing significantly higher bacterial loads in both the spleen and the liver. Moreover, Salmonella-specific Ab production and Ag-specific CD4 T cells were reduced in Sirpα−/− mice compared with wild-type controls. To further characterize the potential mechanism underlying SIRPα-dependent Ag-specific CD4 T cell priming, we demonstrate that lack of SIRPα expression on dendritic cells results in less efficient Ag processing and presentation in vitro. Collectively, these findings demonstrate an indispensable role of SIRPα for protective immunity to Salmonella infection.

DNA primases catalyze the synthesis of oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Prokaryotic primases consist of a zinc-binding domain (ZBD) necessary for recognition of a specific template sequence and a catalytic RNA polymerase domain. Interactions of both domains with the DNA template and ribonucleotides are required for primer synthesis. Five tryptophan residues are dispersed in the primase of bacteriophage T7: Trp-42 in the ZBD and Trp-69, -97, -147, and -255 in the RNA polymerase domain. Previous studies showed that replacement of Trp-42 with alanine in the ZBD decreases primer synthesis, whereas substitution of non-aromatic residues for Trp-69 impairs both primer synthesis and delivery. However, the roles of tryptophan at position 97, 147, or 255 remain elusive. To investigate the essential roles of these residues, we replaced each tryptophan with the structurally similar tyrosine and examined the effect of this subtle alteration on primer synthesis. The substitution at position 42, 97, or 147 reduced primer synthesis, whereas substitution at position 69 or 255 did not. The functions of the tryptophans were further examined at each step of primer synthesis. Alteration of residue 42 disturbed the conformation of the ZBD and resulted in partial loss of the zinc ion, impairing binding to the ssDNA template. Replacement of Trp-97 with tyrosine reduced the binding affinity to NTP and the catalysis step. The replacement of Trp-147 with tyrosine also impaired the catalytic step. Therefore, Trp-42 is important in maintaining the conformation of the ZBD for template binding; Trp-97 contributes to NTP binding and the catalysis step; and Trp-147 maintains the catalysis step.

DNA primases provide oligoribonucleotides for DNA polymerase to initiate lagging strand synthesis. A deficiency in the primase of bacteriophage T7 to synthesize primers can be overcome by genetic alterations that decrease the expression of T7 gene 5.5, suggesting an alternative mechanism to prime DNA synthesis. The product of gene 5.5 (gp5.5) forms a stable complex with the Escherichia coli histone-like protein H-NS and transfer RNAs (tRNAs). The 3′-terminal sequence (5′-ACCA-3′) of tRNAs is identical to that of a functional primer synthesized by T7 primase. Mutations in T7 that suppress the inability of primase reduce the amount of gp5.5 and thus increase the pool of tRNA to serve as primers. Alterations in T7 gene 3 facilitate tRNA priming by reducing its endonuclease activity that cleaves at the tRNA–DNA junction. The tRNA bound to gp5.5 recruits H-NS. H-NS alone inhibits reactions involved in DNA replication, but the binding to gp5.5–tRNA complex abolishes this inhibition.

Despite the importance of Salmonella infections in human and animal health, the target antigens of Salmonella -specific immunity remain poorly defined. We have previously shown evidence for antibody-mediating protection against invasive Salmonellosis in mice and African children. To generate an overview of antibody targeting in systemic Salmonellosis, a Salmonella proteomic array containing over 2,700 proteins was constructed and probed with immune sera from Salmonella -infected mice and humans. Analysis of multiple inbred mouse strains identified 117 antigens recognized by systemic antibody responses in murine Salmonellosis. Importantly, many of these antigens were independently identified as target antigens using sera from Malawian children with Salmonella bacteremia, validating the study of the murine model. Furthermore, vaccination with SseB, the most prominent antigenic target in Malawian children, provided mice with significant protection against Salmonella infection. Together, these data uncover an overlapping immune signature of disseminated Salmonellosis in mice and humans and provide a foundation for the generation of a protective subunit vaccine.

The anti-hyperlipidemic effects of dietary supplementation with sea tangle Laminaria japonica were investigated using an animal model in which normal rats were fed either sea tangle, sea tangle ethanol extract (EE-ST) and sea tangle extracted residue (ER-ST). Total lipid and triglyceride levels in the serum were significantly ( P < 0.05) reduced in rats fed ER-ST at a dose of 200 mg/kg body weight when compared to hyperlipidemic control rats. Significant decreases in serum total cholesterol and low density lipoproteincholesterol levels also occurred in rats fed ER-ST at 200 mg/kg body weight. In addition, the atherosclerosis index and superoxide dismutase in blood lipids were significantly ( P < 0.05) lowered in rats fed ER-ST at 200 mg/kg body weight as compared to control rats. In conclusion, sea tangle and ER-ST exhibited beneficial anti-hyperlipidemic and anti-arteriosclerosis effects.

The replication system of phage T7 provides a model for DNA replication. Biochemical, structural, and single-molecule analyses together provide insight into replisome mechanics. A complex of polymerase, a processivity factor, and helicase mediates leading strand synthesis. Establishment of the complex requires an interaction of the C-terminal tail of the helicase with the polymerase. During synthesis the complex is stabilized by other interactions to provide for a processivity of 5 kilobase (kb). The C-terminal tail also interacts with a distinct region of the polymerase to capture dissociating polymerase to increase the processivity to >17 kb. The lagging strand is synthesized discontinuously within a loop that forms and resolves during each cycle of Okazaki fragment synthesis. The synthesis of a primer as well as the termination of a fragment signal loop resolution.

This paper is to contribute for management and restoration as a basic study of vegetation structure in Aemirang Hill, Nakdongjeongmaek. To verify the characteristics of vegetation structure, we set up 25 plots(100㎡). The result of community analysis was used by TWINSPAN classification. The communities were classified 7 types i.e. Quercus variabilis community, Quercus mongolica community, Pinus densiflora community, Pinus densiflora-Quercus mongolica community, Q. mongolica-Quercus variabilis community, Quercus mongolica-Betula davurica community, Larix leptolepis community. Pinus densiflora community and Pinus densiflora-Quercus mongolica community are progressively transforming a Quercus mongolica community that is called Pinus densiflora next succession stage. Whereas the communities that has dominant Quercus variabilis and Quercus mongolica did not appear a next generation succession tree such as Carpinus laxiflora, Carpinus cordata in the under story and shrub layer. Therefore, Aemirang Hill's ecological succession was to maintain the status condition. Aemirang Hill's diversity index is 0.5738 to 0.8906. It were analyzed that diversity index was lower than other places.

TLR5-deficient mice have been reported to develop spontaneous intestinal inflammation and metabolic abnormalities. However, we report that TLR5-deficient mice from two different animal colonies display no evidence of basal inflammatory disease, metabolic abnormalities, or enhanced resistance to Salmonella infection. In contrast, the absence of TLR5 hindered the initial activation and clonal expansion of intestinal flagellin-specific CD4 T cells following oral Salmonella infection. Together, these data demonstrate that a basal inflammatory phenotype is not a consistent feature of TLR5-deficient mice and document a novel role for TLR5 in the rapid targeting of flagellin by intestinal pathogen-specific CD4 T cells.

This study examined the antidiabetic and antihyperlipidemic activities of an ascidian (Halocynthia roretzi) in rats in vivo. Rats were fed on experimental diet including dried ascidian powder (200 mg/kg body weight) for 4 weeks, and then the triglyceride and total cholesterol levels in blood were analyzed. On the ascidian tunic powder diet, the triglyceride level decreased by more than 20.9% and the total cholesterol content decreased by more than 24.4%. In comparison, the triglyceride and total cholesterol level in the blood of rats fed ascidian meat powder decreased only slightly. Therefore, the ascidian tunic powder might be a healthy food with antidiabetic and antihyperlipidemic effects.

It has been suggested that connexin (Cx) gap junction proteins act as tumor suppressors and green tea has a potential to prevent tumor development, however, the studies on their association with human keratinocytes were rare. We evaluated the effects of a tumor promoter, phorbol-12-myristate-13-acetate (PMA), on the expression of Cxs and gap junction intercellular communication (GJIC) in human keratinocytes (HaCaT cells) and explored the preventive effects of green tea extracts-epicatechin (EC) and epigallocatechin-3-gallate (EGCG). We performed neutral red dye uptake assay to determine the optimal concentrations of PMA, EC, and EGCG for this study and confirmed the expression of Cx mRNAs using RT-PCR. We evaluated GJIC quantitatively using the 'scrape-loading dye transfer (SLDT)' technique after 24-h culture of HaCaT cells treated with agents. To analyze the expression change of Cxs, we also performed Western blot and immunocytochemistry. HaCaT cells were found to express Cx26, Cx30, Cx31, and Cx43, but not Cx29. In 'scrape-loading dye transfer' for functional study for GJIC, EC and EGCG significantly prevented PMA-induced down-regulation of GJIC. Western blot analyses revealed that EC and EGCG prevented down-regulation of Cx26 and Cx43 proteins in HaCaT cells treated with PMA. Immunocytochemistry showed decreased expression and abnormal location of Cx26 and Cx43 in HaCaT cells when treated with PMA, and EC and EGCG inhibited its effect. These results suggest an important role of GJIC played in carcinogenesis involving human keratinocytes and green tea as a useful anticancer diet.

Sea-urchins (Anthocidaris crassispina) are widely distributed in the East Sea of Korea. The aim of this study was to evaluate the hepatoprotective effects of sea-urchin roe on bromobenzene (BB)-induced liver damage in rats. The antioxidative and detoxifying properties of sea-urchin roe in BB-poisoned rat liver was examined by chemical analysis of serum aminotransferase (AST, ALT), glutathione S-transferase (GST), γ-glutamylcystein synthetase, glutathione reductase, epoxide hydrolase, amino-N-demethylase (AD), aniline hydrolase (AH) enzyme activity, as well as lipid peroxide and glutathione contents. Sea-urchin roe inhibited the increase of serum AST, ALT enzyme activity. Increasing lipid peroxide contents and AD and AH activities were significantly decreased in ethanol extract of sea-urchin roe. GST, γ-glutamylcystein synthetase, glutathione reductase and epoxide hydrolase enzyme activities increased in sea-urchin roe-fed group, compared with the BB-treated group. These results suggest that sea-urchin roe facilitates recovery from liver damage by enhancing antioxidative defense mechanisms and hepatic detoxication metabolism.

This study investigated the inhibitory effects of sea mustard on high-fat diet-induced obesity and hyperlipidemia in Sprague-Dawley rats. Sea mustard (Undaria pinnatifida) powder, sea mustard ethanol extract, and sea mustard ethanol-extracted residue were tested. The ethanol extracted residue had the most beneficial anti-hyperlipidemic activity. Alginate in the sea mustard was considered to be the key component. The ethanol-extracted residue of sea mustard also had antioxidant activity, which may be effective in preventing hyperlipidemia by increasing the enzymatic activity of superoxide dismutase, which can remove active oxygen from the bloodstream.