Method to quantify live and dead cells in multi-species oral biofilm by real-time PCR with propidium monoazide

Álvarez, Gerard; Gonzàlez, Marta; Isabal, Sergio; Blanc, Vanessa; León, Rubén

doi:10.1186/2191-0855-3-1

Cited by 183 publications

(141 citation statements)

References 31 publications

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“…In comparison, the newly reported alkali-stable laccases from actinomycetes [12] and Trametes sp. [11] had similar half-lifes (60 minutes) at only 60°C and pH 8, showing the substantial advantage of the characterized bacterial laccases over fungal orthologs if stability is preferred over redox potential.…”

Section: Discussionmentioning

confidence: 88%

Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from Bacillus clausii

2014

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The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ∼0.5–2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (KM) but to pH dependence of catalytic turnover: The kcat of B. clausii cotA was 1 s−1 at pH 6 and 5 s−1 at pH 8 in contrast to 6 s−1 at pH 6 and 2 s−1 at pH 8 for of B. subtilis cotA. Overall, kcat/KM was 10-fold higher for B. subtilis cotA at pHopt. While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500–700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ∼20 minutes half-life at 80°C, less than the ∼50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH∼8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization.

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Section: Discussionmentioning

confidence: 88%

Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from Bacillus clausii

2014

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“…In contrast to E. coli induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) in mid log phase, optimal production of SELPs from E. coli BL21 was obtained by induction at the beginning of the stationary phase [41]. Yields of 25–500 mg/L by shake flask culture [25, 41, 42, 72] and 4.3g/L [43] by bioreactor cultivation were reported for purified SELPs produced in E. coli . However, one of the primary limitations to the successful commercialization of SELPs is still associated with the production of protein on a commercially viable level.…”

Section: Biosynthesis Of Silk-elastin-like Proteinsmentioning

confidence: 99%

Silk-elastin-like protein biomaterials for the controlled delivery of therapeutics

Huang

Rollett

Kaplan

2014

Expert Opinion on Drug Delivery

116

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Introduction Genetically engineered biomaterials are useful for controlled delivery owing to their rational design, tunable structure-function, biocompatibility, degradability and target specificity. Silk-elastin-like proteins (SELPs), a family of genetically engineered recombinant protein polymers, possess these properties. Additionally, given the benefits of combining semicrystalline silk-blocks and elastomeric elastin-blocks, SELPs possess multi-stimuli responsive properties and tenability, thereby, becoming promising candidates for targeted cancer therapeutics delivery and controlled gene release. Areas covered An overview of SELP biomaterials for drug delivery and gene release is provided. Biosynthetic strategies used for SELP production, fundamental physicochemical properties, and self-assembly mechanisms are discussed. The review focuses on sequence-structure-function relationships, stimuli responsive features, and current and potential drug delivery applications. Expert opinion The tunable material properties allow SELPs to be pursued as promising biomaterials for nano-carriers and injectable drug release systems. Current applications of SELPs have focused on thermally-triggered biomaterial formats for the delivery of therapeutics, based on local hyperthermia in tumors or infections. Other prominent controlled release applications of SELPs as injectable hydrogels for gene release have also been pursued. Further biomedical applications that utilize other stimuli to trigger the reversible material responses of SELPs for targeted delivery, including pH, ionic strength, redox, enzymatic stimuli and electric field, are in progress. Exploiting these additional stimuli responsive features will provide a broader range of functional biomaterials for controlled therapeutics release and tissue regeneration.

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“…However, this approach can overestimate the number of viable cells due to the presence of free extracellular DNA (eDNA) (Klein et al, 2012) and DNA derived from dead cells. To avoid quantification of DNA not derived from living cells, samples can be treated with propidium monoazide (PMA) prior to DNA extraction (Alvarez et al, 2013;Kruger et al, 2014;Yasunaga et al, 2013). This molecule enters only membrane-compromised cells, intercalating between bases, and also interacts with eDNA (Nocker et al, 2007;Waring, 1965).…”

Section: Microbiological and Molecular Methodsmentioning

confidence: 99%

Critical review on biofilm methods

Azeredo

Azevedo

Briandet

et al. 2016

Critical Reviews in Microbiology

784

679

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Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms. ARTICLE HISTORY

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Method to quantify live and dead cells in multi-species oral biofilm by real-time PCR with propidium monoazide

Cited by 183 publications

References 31 publications

Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from Bacillus clausii

Characterization of an Alkali- and Halide-Resistant Laccase Expressed in E. coli: CotA from Bacillus clausii

Silk-elastin-like protein biomaterials for the controlled delivery of therapeutics

Critical review on biofilm methods

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