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Pesquisa : D01.625.100 [Categoria DeCS]
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  1 / 9722 MEDLINE  
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PMID:29073460
Autor:Scariot MC; Venturelli GL; Prudêncio ES; Arisi ACM
Endereço:CAL CCA UFSC, Food Science and Technology Department, Federal University of Santa Catarina, Rod. Admar Gonzaga, 1346, 88034-001 Florianópolis, SC, Brazil.
Título:Quantification of Lactobacillus paracasei viable cells in probiotic yoghurt by propidium monoazide combined with quantitative PCR.
Fonte:Int J Food Microbiol; 264:1-7, 2018 Jan 02.
ISSN:1879-3460
País de publicação:Netherlands
Idioma:eng
Resumo:Propidium monoazide (PMA) coupled with qPCR has been successfully used for specific quantification of viable bacteria cells in diverse matrices food. The present study aimed to develop PMA-qPCR assay for quantification of Lactobacillus paracasei viable cells in probiotic yoghurt. L. paracasei grown in culture medium was submitted to heat treatment at 60°C for different periods of time and probiotic yoghurt containing L. paracasei were prepared and stored at 4°C for 30days. The viable cells were quantified using qPCR and PMA-qPCR assays targeting tuf gene and also by plate counting. Standard curves were prepared and mean efficiency obtained was 94% and 96% (R >0.98) to L. paracasei in culture medium and probiotic yoghurt stored one day, respectively. The limit of detection (LOD) for both samples was 10 genome copies, corresponding to 32.1pg of DNA. For viable cells quantification, standard curves Cq versus log CFU were plotted using mean CFU by plate counting of L. paracasei grown in culture medium and probiotic yoghurt. Results obtained for L. paracasei heat-treated cells were concordant by PMA-qPCR and plate count, CFU decreased as the heat treatment time increased, while qPCR count remained constant. L. paracasei enumerations obtained by qPCR for probiotic yoghurt stored one day and 30days were higher than enumerations by PMA-qPCR for the same samples. The plate count values were similar to CFU values obtained by PMA-qPCR. These results showed that PMA-qPCR is a powerful approach compared with culture-dependent methods for quantification of L. paracasei viable cells in yoghurt. PMA-qPCR allowed reliable obtained results much faster than plate counting.
Tipo de publicação: JOURNAL ARTICLE
Nome de substância:0 (Azides); 0 (DNA, Bacterial); 0 (propidium monoazide); 36015-30-2 (Propidium)


  2 / 9722 MEDLINE  
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PMID:29248298
Autor:Chatkewitz LE; Halonski JF; Padilla MS; Young DD
Endereço:Department of Chemistry, College of William & Mary, P.O. Box 8795, Williamsburg, VA 23187 USA.
Título:Investigation of copper-free alkyne/azide 1,3-dipolar cycloadditions using microwave irradiation.
Fonte:Bioorg Med Chem Lett; 28(2):81-84, 2018 01 15.
ISSN:1464-3405
País de publicação:England
Idioma:eng
Resumo:The prevalence of 1,3-dipolar cycloadditions of azides and alkynes within both biology and chemistry highlights the utility of these reactions. However, the use of a copper catalyst can be prohibitive to some applications. Consequently, we have optimized a copper-free microwave-assisted reaction to alleviate the necessity for the copper catalyst. A small array of triazoles was prepared to examine the scope of this approach, and the methodology was translated to a protein context through the use of unnatural amino acids to demonstrate one of the first microwave-mediated bioconjugations involving a full length protein.
Tipo de publicação: JOURNAL ARTICLE
Nome de substância:0 (Alkynes); 0 (Azides); 0 (Triazoles)


  3 / 9722 MEDLINE  
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PMID:28448459
Autor:Bonetta S; Pignata C; Bonetta S; Meucci L; Giacosa D; Marino E; Gilli G; Carraro E
Endereço:Department of Public Health and Pediatrics, University of Torino, P.zza Polonia 94, 10126 Torino, Italy. sara.bonetta@unito.it.
Título:Viability of Legionella pneumophila in Water Samples: A Comparison of Propidium Monoazide (PMA) Treatment on Membrane Filters and in Liquid.
Fonte:Int J Environ Res Public Health; 14(5), 2017 04 27.
ISSN:1660-4601
País de publicação:Switzerland
Idioma:eng
Resumo:is a ubiquitous microorganism widely distributed in aquatic environments and can cause Legionellosis in humans. A promising approach to detect viable cells in water samples involves the use of quantitative polymerase chain reaction (qPCR) in combination with photoactivatable DNA intercalator propidium monoazide (PMA). However, the PMA efficiency could be different depending on the experimental conditions used. The aim of this study was to compare two PMA exposure protocols: (A) directly on the membrane filter or (B) in liquid after filter washing. The overall PMA-induced qPCR means reductions in heat-killed cells were 2.42 and 1.91 log units for exposure protocols A and B, respectively. A comparison between the results obtained reveals that filter exposure allows a higher PMA-qPCR signal reduction to be reached, mainly at low concentrations ( < 0.05). This confirms the potential use of this method to quantify in water with low contamination.
Tipo de publicação: COMPARATIVE STUDY; JOURNAL ARTICLE
Nome de substância:0 (Azides); 0 (Coloring Agents); 0 (Membranes, Artificial); 0 (propidium monoazide); 36015-30-2 (Propidium)


  4 / 9722 MEDLINE  
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PMID:29032906
Autor:Jäger T; Alexander J; Kirchen S; Dötsch A; Wieland A; Hiller C; Schwartz T
Endereço:Karlsruhe Institute of Technology (KIT) - Campus North, Institute of Functional Interfaces (IFG), Department of Bioengineering and Biosystems, P.O. Box 3640, 76021 Karlsruhe, Germany.
Título:Live-dead discrimination analysis, qPCR assessment for opportunistic pathogens, and population analysis at ozone wastewater treatment plants.
Fonte:Environ Pollut; 232:571-579, 2018 Jan.
ISSN:1873-6424
País de publicação:England
Idioma:eng
Resumo:In respect to direct and indirect water reuse, the microbiological quality of treated wastewater is highly important. Conventional wastewater treatment plants are normally not equipped with advanced technologies for the elimination of bacteria. Molecular biology analyses were combined with live-dead discrimination analysis of wastewater population using Propidium monoazide (PMA) to study population shifts during ozonation (1 g ozone/g DOC) at a municipal wastewater treatment plant. Escherichia coli, enterococci, and Pseudomonas aeruginosa were quantified by polymerase chain reaction (qPCR) and the whole wastewater population was analyzed by metagenomic sequencing. The PMA-qPCR experiments showed that the abundances of P. aeruginosa didn't change by ozone treatment, whereas a reduction was observed for E. coli and enterococci. Results comparing conventional cultivation experiments with PMA-qPCR underlined the presence of viable but not culturable cells (VBNC) and their regrowth potential after ozone treatment. Illumina HiSeq sequencing results with and without PMA treatment demonstrated high population similarities in water samples originating from ozone inflow sampling sides. Upon using PMA treatment after ozonation, population shifts became visible and also underlined the importance of PMA treatment for the evaluation of elimination and selection processes during ozonation at WWTPs. Amongst a number of 14 most abundant genera identified in the inflow samples, 9 genera were found to be reduced, whereas 4 genera increased in relative abundance and 1 genus almost remained constant. The strongest increase in relative abundance after ozonation was detected for Oscillatoria spp., Microcoleus spp. and Nitrospira spp. Beside this, a continuous release of Pseudomonas spp. (including P. aeruginosa) to the downstream receiving body was confirmed. Regrowth experiments demonstrated a high prevalence of P. aeruginosa as part of the surviving bacterial population. Summing up, molecular biology analyses in combination with live-dead discrimination are comprehensive methods to evaluate the elimination processes targeting specific species and/or whole microbial populations.
Tipo de publicação: JOURNAL ARTICLE
Nome de substância:0 (Azides); 0 (Waste Water); 0 (propidium monoazide); 36015-30-2 (Propidium); 66H7ZZK23N (Ozone)


  5 / 9722 MEDLINE  
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PMID:27778291
Autor:Soriano GP; Overkleeft HS; Florea BI
Endereço:Bio-organic Synthesis Group, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
Título:Two-Step Activity-Based Protein Profiling with the Proteasome System as Model of Study.
Fonte:Methods Mol Biol; 1491:205-215, 2017.
ISSN:1940-6029
País de publicação:United States
Idioma:eng
Resumo:Activity-based protein profiling (ABPP) is a method to highlight enzymatic activities in a biological sample, which uses chemical probes that react covalently with the catalytic nucleophile of the enzyme. To circumvent disadvantages associated with the presence of reporter tags on chemical probes, the probe is equipped with a ligation handle to which the reporter can be reacted at the desired time and place in the ABPP workflow. This chapter demonstrates the power of a triple bioorthogonal ligation strategy which addresses the three activities of the proteasome: the ß5-subunit selective norbornene-tagged probe is reacted with fluorescent tetrazine, the ß1-selective azide-functionalized probe was addressed with a biotinylated phosphine, followed by an alkyne-substituted pan-reactive probe to label the remaining ß2 activity to which an azide-coupled fluorophore was ligated. The result of the triple ligation was similar to each reaction performed separately demonstrating the value of the triple ligation strategy for a single experiment.
Tipo de publicação: JOURNAL ARTICLE
Nome de substância:0 (Azides); 0 (Molecular Probes); 0 (Proteins); EC 3.4.25.1 (Proteasome Endopeptidase Complex)


  6 / 9722 MEDLINE  
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PMID:29188993
Autor:Santiana JJ; Sui B; Gomez N; Rouge JL
Endereço:Department of Chemistry, University of Connecticut , Storrs, Connecticut 06269, United States.
Título:Programmable Peptide-Cross-Linked Nucleic Acid Nanocapsules as a Modular Platform for Enzyme Specific Cargo Release.
Fonte:Bioconjug Chem; 28(12):2910-2914, 2017 Dec 20.
ISSN:1520-4812
País de publicação:United States
Idioma:eng
Resumo:Herein we describe a modular assembly strategy for photo-cross-linking peptides into nucleic acid functionalized nanocapsules. The peptides embedded within the nanocapsules form discrete nanoscale populations capable of gating the release of molecular and nanoscale cargo using enzyme-substrate recognition as a triggered release mechanism. Using photocatalyzed thiol-yne chemistry, different peptide cross-linkers were effectively incorporated into the nanocapsules and screened against different proteases to test for degradation specificity both in vitro and in cell culture. By using a combination of fluorescence assays, confocal and TEM microscopy, the particles were shown to be highly specific for their enzyme targets, even between enzymes of similar protease classes. The rapid and modular nature of the assembly strategy has the potential to be applied to both intracellular and extracellular biosensing and drug delivery applications.
Tipo de publicação: JOURNAL ARTICLE
Nome de substância:0 (Azides); 0 (Drug Carriers); 0 (Nanocapsules); 0 (Nucleic Acids); 0 (Peptides); 0 (Sulfhydryl Compounds); 7440-57-5 (Gold); 91I69L5AY5 (Enflurane); EC 3.4.24.35 (Matrix Metalloproteinase 9)


  7 / 9722 MEDLINE  
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PMID:27779387
Autor:Ngambenjawong C; Pineda JM; Pun SH
Endereço:Department of Bioengineering and Molecular Engineering and Sciences Institute, University of Washington , Seattle, Washington 98195, United States.
Título:Engineering an Affinity-Enhanced Peptide through Optimization of Cyclization Chemistry.
Fonte:Bioconjug Chem; 27(12):2854-2862, 2016 Dec 21.
ISSN:1520-4812
País de publicação:United States
Idioma:eng
Resumo:Peptide cyclization is a strategy used to improve stability and activity of peptides. The most commonly used cyclization method is disulfide bridge formation of cysteine-containing peptides, as is typically found in nature. Over the years, an increasing number of alternative chemistries for peptide cyclization with improved efficiency, kinetics, orthogonality, and stability have been reported. However, there has been less appreciation for the opportunity to fine-tune peptide activity via the diverse chemical entities introduced at the site of linkage by different cyclization strategies. Here, we demonstrate how cyclization optimization of an M2 "anti-inflammatory" macrophage-binding peptide (M2pep) resulted in a significant increase in binding affinity of the optimized analog to M2 macrophages while maintaining binding selectivity compared to M1 "pro-inflammatory" macrophages. In this study, we report synthesis and evaluation of four cyclic M2pep(RY) analogs with diverse cyclization strategies: (1) Asp-[amide]-Lys, (2) azido-Lys-[triazole(copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC))]-propargyl-Gly, (3) Cys-[decafluorobiphenyl (DFBP)]-Cys, and (4) Cys-[decafluorobiphenyl sulfone (DFS)]-Cys, whereby the chemical entity or linker at the linkage site is shown in the square bracket and is between the residues involved in cyclization. These peptides are compared to a disulfide-cyclized M2pep(RY) that we previously reported as a serum-stable, affinity-enhanced analog to the original linear M2pep. DFBP-cyclized M2pep(RY) exhibits the highest binding activity to M2 macrophages with apparent dissociation constant (K ) about 2.03 µM compared to 36.3 µM for the original disulfide-cyclized M2pep(RY) and 220 µM for the original linear peptide. DFS-cyclized M2pep(RY) also binds more strongly than the original cyclized analog, whereas amide- and triazole-cyclized M2pep(RY) analogs bind less strongly. We verified that DFBP alone has negligible binding to M2 macrophages and the incorporation of diphenylalanine to the original sequence improves binding activity at the expense of solubility and increased toxicity. In conclusion, we report development of cyclic M2pep(RY) analogs with diverse cyclization strategies leading to the discovery of DFBP-cyclized M2pep(RY) with enhanced M2 macrophage-binding activity.
Tipo de publicação: JOURNAL ARTICLE
Nome de substância:0 (Azides); 0 (Peptides); 6SO6U10H04 (Biotin)


  8 / 9722 MEDLINE  
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PMID:27771087
Autor:Zhou B; Chen B; Wu X; Li F; Yu P; Aguilar ZP; Wei H; Xu H
Endereço:State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, 330047, PR China.
Título:A new application of a sodium deoxycholate-propidium monoazide-quantitative PCR assay for rapid and sensitive detection of viable Cronobacter sakazakii in powdered infant formula.
Fonte:J Dairy Sci; 99(12):9550-9559, 2016 Dec.
ISSN:1525-3198
País de publicação:United States
Idioma:eng
Resumo:A rapid, reliable, and sensitive method for the detection of Cronobacter sakazakii, a common foodborne pathogen that may cause serious neonatal disease, has been developed. In this study, a rapid real-time quantitative PCR (qPCR) assay combined with sodium deoxycholate (SD) and propidium monoazide (PMA) was developed to detect C. sakazakii contamination in powdered infant formula (PIF). This method could eliminate the interference from dead or injured bacteria. Optimization studies indicated that SD and PMA at 0.08% (wt/vol) and 5µg/mL, respectively, were the most appropriate. In addition, qPCR, PMA-qPCR, SD-PMA-qPCR, and plate count assays were used to account for the number of viable bacteria in cell suspensions that were exposed to a 55°C water bath at different length of time. As a result, the viable number by PMA-qPCR showed significantly higher than of the number from SD-PMA-qPCR or plate counts. The number of viable bacteria was consistent between SD-PMA-qPCR and traditional plate counts, which indicated that SD treatment could eliminate the interference from dead or injured cells. Using the optimized parameters, the limit of detection with the SD-PMA-qPCR assay was 3.3×10 cfu/mL and 4.4×10 cfu/g in pure culture and in spiked PIF, respectively. A similar detection limit of 5.6×10 cfu/g was obtained in the presence of the Staphylococcus aureus (10 cfu/mL). The combined SD-PMA-qPCR assay holds promise for the rapid detection of viable C. sakazakii in PIF.
Tipo de publicação: JOURNAL ARTICLE
Nome de substância:0 (Azides); 005990WHZZ (Deoxycholic Acid); 36015-30-2 (Propidium)


  9 / 9722 MEDLINE  
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PMID:27771082
Autor:Shao Y; Wang Z; Bao Q; Zhang H
Endereço:College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi' an, Shaanxi, P. R. China, 710119.
Título:Application of propidium monoazide quantitative real-time PCR to quantify the viability of Lactobacillus delbrueckii ssp. bulgaricus.
Fonte:J Dairy Sci; 99(12):9570-9580, 2016 Dec.
ISSN:1525-3198
País de publicação:United States
Idioma:eng
Resumo:In this study, a combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) was used to develop a method to determine the viability of cells of Lactobacillus delbrueckii ssp. bulgaricus ND02 (L. bulgaricus) that may have entered into a viable but nonculturable state. This can happen due to its susceptibility to cold shock during lyophilization and storage. Propidium monoazide concentration, PMA incubation time, and light exposure time were optimized to fully exploit the PMA-qPCR approach to accurately assess the total number of living L. bulgaricus ND02. Although PMA has little influence on living cells, when concentrations of PMA were higher than 30µg/mL the number of PCR-positive living bacteria decreased from 10 to 10 cfu/mL in comparison with qPCR enumeration. Mixtures of living and dead cells were used as method verification samples for enumeration by PMA-qPCR, demonstrating that this method was feasible and effective for distinguishing living cells of L. bulgaricus when mixed with a known number of dead cells. We suggest that several conditions need to be studied further before PMA-qPCR methods can be accurately used to distinguish living from dead cells for enumeration under more realistic sampling situations. However, this research provides a rapid way to enumerate living cells of L. bulgaricus and could be used to optimize selection of cryoprotectants in the lyophilization process and develop technologies for high cell density cultivation and optimal freeze-drying processes.
Tipo de publicação: JOURNAL ARTICLE
Nome de substância:0 (Azides); 36015-30-2 (Propidium)


  10 / 9722 MEDLINE  
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PMID:28457755
Autor:Kitamura Y; Asakura R; Terazawa K; Shibata A; Ikeda M; Kitade Y
Endereço:Department of Biomolecular Science, Graduate School of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Título:Nucleobase azide-ethynylribose click chemistry contributes to stabilizing oligonucleotide duplexes and stem-loop structures.
Fonte:Bioorg Med Chem Lett; 27(12):2655-2658, 2017 06 15.
ISSN:1464-3405
País de publicação:England
Idioma:eng
Resumo:The formation of 1,4-disubstituted 1,2,3-triazoles through copper-catalyzed azide-alkyne cycloaddition (CuAAC) in oligonucleotides bearing 1-deoxy-1-ethynyl-ß-d-ribofuranose (R ) can have a positive impact on the stability of oligonucleotide duplexes and stem-loop structures.
Tipo de publicação: JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
Nome de substância:0 (Azides); 0 (Deoxyribonucleotides); 0 (Triazoles); 789U1901C5 (Copper)



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