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Simple and rapid real-time monitoring of LPL activity in vitro.

ABSTRACT: Since elevated plasma triglycerides are an independent risk factor for cardiovascular diseases, lipoprotein lipase (LPL) is an interesting target for drug development. However, investigation of LPL remains challenging, as most of the commercially available assays are limited to the determination of LPL activity. Thus, we focused on the evaluation of a simple in vitro real-time fluorescence assay for the measurement of LPL activity that can be combined with additional cell or molecular biological assays in the same cell sample. Our procedure allows for a more comprehensive characterization of potential regulatory compounds targeting the LPL system. The presented assay procedure provides several advantages over currently available commercial in vitro LPL activity assays:1.12-well cell culture plate design for the simultaneous investigation of up to three different compounds of interest (including all assay controls).2.24 h real-time acquisition of LPL activity for the identification of the optimal time point for further measurements.3.Measurement of LPL activity can be supplemented by additional cell or molecular biological assays in the same cell sample.

SUBMITTER: Kluge S

PROVIDER: S-EPMC7132154 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Simple and rapid real-time monitoring of LPL activity <i>in vitro</i>.

Kluge Stefan S Boermel Lisa L Schubert Martin M Lorkowski Stefan S

MethodsX 20200317

Since elevated plasma triglycerides are an independent risk factor for cardiovascular diseases, lipoprotein lipase (LPL) is an interesting target for drug development. However, investigation of LPL remains challenging, as most of the commercially available assays are limited to the determination of LPL activity. Thus, we focused on the evaluation of a simple <i>in vitro</i> real-time fluorescence assay for the measurement of LPL activity that can be combined with additional cell or molecular bio ...[more]

PMID: 32274337

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Project description:BackgroundPrecise control of gene expression is essential to synchronize plant development with the environment. In perennial plants, transcriptional regulation remains poorly understood, mainly due to the long time required to perform functional studies. Transcriptional reporters based on luciferase have been useful to study circadian and diurnal regulation of gene expression, both by transcription factors and chromatin remodelers. The high mobility group proteins are considered transcriptional chaperones that also modify the chromatin architecture. They have been found in several species, presenting in some cases a circadian expression of their mRNA or protein.ResultsTransactivation experiments have been shown as a powerful and fast method to obtain information about the potential role of transcription factors upon a certain reporter. We designed and validated a luciferase transcriptional reporter using the 5' sequence upstream ATG of Populus tremula × alba LHY2 gene. We showed the robustness of this reporter line under long day and continuous light conditions. Moreover, we confirmed that pPtaLHY2::LUC activity reproduces the accumulation of PtaLHY2 mRNA. We performed transactivation studies by transient expression, using the reporter line as a genetic background, unraveling a new function of a high mobility group protein in poplar, which can activate the PtaLHY2 promoter in a gate-dependent manner. We also showed PtaHMGB2/3 needs darkness to produce that activation and exhibits an active degradation after dawn, mediated by the 26S proteasome.ConclusionsWe generated a stable luciferase reporter poplar line based on the circadian clock gene PtaLHY2, which can be used to investigate transcriptional regulation and signal transduction pathway. Using this reporter line as a genetic background, we established a methodology to rapidly assess potential regulators of diurnal and circadian rhythms. This tool allowed us to demonstrate that PtaHMGB2/3 promotes the transcriptional activation of our reporter in a gate-dependent manner. Moreover, we added new information about the PtaHMGB2/3 protein regulation along the day. This methodology can be easily adapted to other transcription factors and reporters.

Project description:Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for both acute and chronic infections in humans. In particular, its ability to form biofilm, on biotic and abiotic surfaces, makes it particularly resistant to host's immune defenses and current antibiotic therapies as well. Innovative antimicrobial materials, like hydrogel, silver salts or nanoparticles have been used to cover new generation catheters with promising results. Nevertheless, biofilm remains a major health problem. For instance, biofilm produced onto endotracheal tubes (ETT) of ventilated patients plays a relevant role in the onset of ventilation-associated pneumonia. Most of our knowledge on Pseudomonas aeruginosa biofilm derives from in vitro studies carried out on abiotic surfaces, such as polystyrene microplates or plastic materials used for ETT manufacturing. However, these approaches often provide underestimated results since other parameters, in addition to bacterial features (i.e. shape and material composition of ETT) might strongly influence biofilm formation.We used an already established biofilm development assay on medically-relevant foreign devices (CVC catheters) by a stably transformed bioluminescent (BLI)-Pseudomonas aeruginosa strain, in order to follow up biofilm formation on ETT by bioluminescence detection. Our results demonstrated that it is possible: i) to monitor BLI-Pseudomonas aeruginosa biofilm development on ETT pieces in real-time, ii) to evaluate the three-dimensional structure of biofilm directly on ETT, iii) to assess metabolic behavior and the production of microbial virulence traits of bacteria embedded on ETT-biofilm.Overall, we were able to standardize a rapid and easy-to-perform in vitro model for real-time monitoring Pseudomonas aeruginosa biofilm formation directly onto ETT pieces, taking into account not only microbial factors, but also ETT shape and material. Our study provides a rapid method for future screening and validation of novel antimicrobial drugs as well as for the evaluation of novel biomaterials employed in the production of new classes of ETT.

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Simple and rapid real-time monitoring of LPL activity in vitro.

Publications

Simple and rapid real-time monitoring of LPL activity <i>in vitro</i>.

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