High-Throughput Analysis of Protein with Tandem Fluorescent Protein Timers

Tandem fluorescent protein timers (tFTs) are versatile reporters of protein dynamics. A tFT consists of two fluorescent proteins with different maturation kinetics and provides a ratiometric readout of protein age, which can be exploited to follow intracellular trafficking, inheritance and turnover of tFT-tagged proteins. Here, we detail a protocol for high-throughput analysis of protein turnover with tFTs in yeast using fluorescence measurements of ordered colony arrays. We describe guidelines on optimization of experimental design with regard to the layout of colony arrays, growth conditions, and instrument choice. Combined with semi-automated genetic crossing using synthetic genetic array (SGA) methodology and high-throughput protein tagging with SWAp-Tag (SWAT) libraries, this approach can be used to compare protein turnover across the proteome and to identify regulators of protein turnover genome-wide.

Highly efficient fluorescence resonance energy transfer in co-encapsulated BODIPY nanoparticles

Fluorescence resonance energy transfer (FRET) is a powerful tool used in a wide range of applications due to its high sensitivity and many other advantages. Co-encapsulation of a donor and an acceptor in nanoparticles is a useful strategy to bring the donor-acceptor pair in proximity for FRET. A highly efficient FRET system based on BODIPY-BODIPY (BODIPY: boron-dipyrromethene) donor-acceptor pair in nanoparticles was synthesized. Nanoparticles were formed by co-encapsulating a green emitting BODIPY derivative (FRET donor, lmax = 501 nm) and a red emitting BODIPY derivative (FRET acceptor, lmax = 601 nm) in an amphiphilic polymer using the precipitation method. Fluorescence measurements of encapsulated BODIPY in water following 501 nm excitation caused a 3.6 fold enhancement of the acceptor BODIPY emission at 601 nm indicating efficient energy transfer between the green emitting donor BODIPY and the red emitting BODIPY acceptor with a 100 nm Stokes shift. The calculated FRET efficiency was 96.5%. Encapsulated BODIPY derivatives were highly stable under our experimental conditions.

The Influence of the Type of Dry-Cured Italian PDO Ham on Cathepsin B Activity Trend during Processing

Cathepsin B activity was measured during processing in hams originating from the main Italian prosciutto PDOs: Parma, San Daniele and Toscano. Sixty-five heavy pig thighs, from sixty-five Italian large white x Italian Landrace pigs bred and slaughtered in the same conditions were considered. Five thighs represented the post-mortem control time. The other 60 were distributed one plant per PDO, following a balanced plan. The thighs were sampled at the biceps femoris in groups of four per plant in the following ripening phases: salting, resting, drying, greasing, end of curing. The activity of the Cathepsin B (U/g protein) was determined by means of fluorescence measurements. The Cathepsin B ripening trend of the various PDOs was significantly different, particularly during the initial and mid-curing stage. This activity correlates with the proteolysis index through a PDO dependent pattern, indicating that different processing conditions can influence the quality of prosciutto, since they determine its biochemical development.

Keratinocyte cellular damage induced by pesticide doses below the cytotoxic level evidenced by electrical impedance and broadband dielectric spectroscopy

Cellular response of a normal human keratinocyte cell line exposed to non-cytotoxic doses of a deltamethrin-based pesticide was investigated by means of two different electrical impedance data spectroscopy approaches: Nyquist plot and broadband dielectric spectroscopy. The measurements have shown that the membrane capacity increases with pesticide concentration and this facilitates the electric current through cell membranes. Furthermore, the impedance of the extracellular matrix also increases with pesticide concentration, thus reducing the electric current outside the cell. Dielectric permittivity changes in the cellular samples at frequency larger than 100 Hz. Fluorescence measurements emphasized an increase of neutral membrane lipids as consequence of the pesticide exposure. Comparison of fluorescence response of pesticide exposed cells with the control ones showed a time increase of the emission intensity, suggesting the existence of a membrane lipid response aimed at repairing of the cell damage due to pesticide exposure. Therefore, both the spectroscopic techniques have demonstrated to be potential means to investigate the response to cell stress and damage. This opens up new possibilities in the early diagnosis of cellular modifications related to pesticides exposure of cells.

Assessment of real-time bioaerosol particle counters using reference chamber experiments

This study presents the first reference calibrations of three commercially available bioaerosol detectors. The Droplet Measurement Technologies WIBS-NEO (new version of the wideband integrated bioaerosol spectrometer), Plair Rapid-E, and Swisens Poleno were compared with a primary standard for particle number concentrations at the Federal Institute for Metrology (METAS). Polystyrene (PSL) spheres were used to assess absolute particle counts for diameters from 0.5 to 10 µm. For the three devices, counting efficiency was found to be strongly dependent on particle size. The results confirm the expected detection range for which the instruments were designed. While the WIBS-NEO achieves its highest efficiency with smaller particles, e.g. 90 % for 0.9 µm diameter, the Plair Rapid-E performs best for larger particles, with an efficiency of 58 % for particles with a diameter of 10 µm. The Swisens Poleno is also designed for larger particles but operates well from 2 µm. However, the exact counting efficiency of the Poleno could not be evaluated as the cut-off diameter range of the integrated concentrator unit was not completely covered. In further experiments, three different types of fluorescent particles were tested to investigate the fluorescent detection capabilities of the Plair Rapid-E and the Swisens Poleno. Both instruments showed good agreement with the reference data. While the challenge to produce known concentrations of larger particles above 10 µm or even fresh pollen particles remains, the approach presented in this paper provides a potential standardised validation method that can be used to assess counting efficiency and fluorescence measurements of automatic bioaerosol monitoring devices.

A Luminescent, Water-Soluble Ir(III) Complex as a Potential Photosensitizer for Two-Photon Photodynamic Therapy

This work reports the study of two-photon induced properties of a highly luminescent cyclometalated Ir(III) complex, [Ir(ppy)2(en)] OOCCH3 (1), ppy = 2-phenylpyridine, en = ethylenediamine. Steady-state and time-resolved fluorescence measurements were performed by exciting 1 at the biologically relevant wavelength of 800 nm, whereas, the generation of singlet oxygen (1O2) was evaluated using 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ABDA) as a detection probe. Preliminary in vitro experiments with U87-MG cells were performed, showing the potential of this compound as a two-photon photodynamic therapy (2P-PDT) agent at NIR wavelengths.

Screening for Optimal Liposome Preparation Conditions by Using Dual Centrifugation and Time-Resolved Fluorescence Measurements

Dual centrifugation (DC) is a novel in-vial homogenization technique for the preparation of liposomes in small batch sizes under gentle and sterile conditions which allows encapsulation efficiencies (EE) for water soluble compounds of >50%. Since liposome size, size distribution (PDI), and EE depend on the lipid concentration used in the DC process, a screening method to find optimal lipid concentrations for a defined lipid composition was developed. Four lipid mixtures consisting of cholesterol, hydrogenated or non-hydrogenated egg PC, and/or PEG-DSPE were screened and suitable concentration ranges could be identified for optimal DC homogenization. In addition to the very fast and parallel liposome preparation of up to 40 samples, the screening process was further accelerated by the finding that DC generates homogeneously mixed liposomes from a macroscopic lipid mixture without the need to initially prepare a molecularly mixed lipid film from an organic solution of all components. This much simpler procedure even works for cholesterol containing lipid blends, which could be explained by a nano-milling of the cholesterol crystals during DC homogenization. Furthermore, EE determination was performed by time-resolved fluorescence measurements of calcein-loaded liposomes without removing the non-entrapped calcein. The new strategy allows the rapid characterization of a certain lipid composition for the preparation of liposomes within a working day.

A new replication medium enables a rapid identification of φx-174 virus by synchronous fluorescence of Tryptophan.

Development of rapid methods for identification of bacteriophages based on their intrinsic fluorescence is challenging. Pure bacteriophages may be detected based on the strong fluorescence of the amino acid Tryptophan that exist in their proteins. Nevertheless, Tryptophan is a molecule that also exist in high quantities in the bacterial hosts and their cultivation media. In this work, we show that simple separation of the bacteriophage φx-174 from its E.coli host (grown on standard cultivation medium) by filtration is not sufficient for its identification based on the intrinsic fluorescence of its Tryptophan content. This is mostly because of the tryptophan residues that derive from the cultivation medium. We fabricate a new cultivation medium that does not have any significant fluorescence overlap with Tryptophan. By utilization of this new cultivation medium, we can identify φx174 based on the spectral fingerprint of its intrinsic Tryptophan content by synchronous fluorescence measurements.

The quantitative contribution of different Photosystem II compartments to non-photochemical quenching in Arabidopsis

Excess excitation energy in the light-harvesting antenna of Photosystem II (PSII) can cause irreversible damage to the photosynthetic apparatus. In periods of high light intensity, a feedback mechanism known as non-photochemical quenching (NPQ), induces the formation of quenchers which can safely dissipate excess excitation energy as heat. Although quenchers have been identified in more than one compartment of the PSII supercomplex, there is currently no quantitative description of how much NPQ is occurring at each of these locations. Here, we perform time-resolved fluorescence measurements on WT and antenna mutants lacking LHCII (NoL) and all peripheral antenna (Ch1 and Ch1lhcb5). By combining the results with those of steady-state fluorescence experiments we are able to estimate the intrinsic rate of NPQ for each plant and each PSII compartment. It is concluded that 60-70% of quenching occurs in LHCII, 15-20% in the minor antenna and 15-20% in the PSII core.