Efficacy of violet-blue light to inactive microbial growth.

The increase in health care-associated infections and antibiotic resistance has led to a growing interest in the search for innovative technologies to solve these problems. In recent years, the interest of the scientific community has focused on violet-blue light at 405 nm (VBL405). This study aimed to assess the VBL405 efficiency in reducing microbial growth on surfaces and air. This descriptive study run between July and October 2020. Petri dishes were contaminated with P. aeruginosa, E. coli, S. aureus, S. typhimurium, K. pneumoniae and were placed at 2 and 3 m from a LED light source having a wavelength peak at 405 nm and an irradiance respectively of 967 and 497 µW/cm2. Simultaneously, the air in the room was sampled for 5 days with two air samplers (SAS) before and after the exposition to the VBL405 source. The highest microbial reduction was reached 2 m directly under the light source: S. typhimurium (2.93 log10), K. pneumoniae (2.30 log10), S. aureus (3.98 log10), E. coli (3.83 log10), P. aeruginosa (3.86 log10). At a distance of 3 m from the light source, the greatest reduction was observed for S. aureus (3.49 log10), and P. aeruginosa (3.80 log10). An average percent microbial reduction of about 70% was found in the sampled air after 12 h of exposure to VBL405. VBL405 has proven to contrast microbial growth on the plates. Implementing this technology in the environment to provide continuous disinfection and to control microbial presence, even in the presence of people, may be an innovative solution.

Expression, purification and characterisation of the recombinant possum lipocalin vulpeculin.

BACKGROUND: Lipocalins are a large family of proteins, which possess a highly conserved eight-stranded antiparallel beta-barrel structure as distinctive trait. This family includes Major Urinary Proteins (MUPs) from rats and mouse, studied for their role in urinary protein-mediated chemosignalling. Vulpeculin has been identified as the most abundant protein in the urine of the common brushtail possum, Trichosurus vulpecula. On the basis of high similarity with other MUPS, we hypothesised that vulpeculin might have a role in possum chemosignalling and investigated its stability and binding ability. METHODS: We expressed and purified vulpeculin using an E.coli-based system and confirmed correct folding by circular dichroism (CD) spectroscopy. Thermal stability was studied by CD and binding properties were investigated using two optical probes N-phenyl-naphthylamine (NPN) and 8-anilino-1-naphthalene sulphonic acid (ANS). RESULTS: CD revealed a secondary structure typical of a predominantly ß-sheet protein, consistent with the beta barrel structure of the lipocalin family. Vulpeculin showed a high level of thermostability, as assessed by CD, exhibiting a small shift in the secondary structure even at 95 °C. Binding assays indicated that vulpeculin cannot accommodate the NPN ligand but can bind ANS. CONCLUSION: The urinary secretion, high degree of sequence similarity with other lipocalins, its beta sheet structure assessed by CD and potential to bind hydrophobic ligands in the hydrophobic cavity or an external hydrophobic pocket, suggest vulpeculin may be involved in possum chemosignalling. GENERAL SIGNIFICANCE: This work represents a first step towards the further investigation of the newly discovered lipocalin and its role in possum chemosignalling.

Selection and characterization of DNA aptamers for the rat major urinary protein 13 (MUP13) as selective biorecognition elements for sensitive detection of rat pests.

Among invasive mammalian predators, rats represent a major threat, endangering ecosystem functioning worldwide. After rat-control operations, detecting their continued presence or reinvasion requires more sensitive and lower cost detection technologies. Here, we develop a new sensing paradigm by using a specific rat urine biomarker (MUP13) to unambiguously signal the presence of rats. As the first step towards a new remote surveillance technology, aptamers were selected to MUP13 using the Flu-Mag SELEX method. Six aptamer candidates were initially screened by dot blot and two of them (Apt-2.5 and Apt-1.4) exhibited high affinity and specificity. Both aptamers were further characterized by bead-based assay to confirm affinity and selectivity. The lead aptamer candidates were then applied to fluorescence anisotropy (FA) and surface plasmon resonance (SPR)-based biosensor platforms, showing dissociation constants in the nanomolar range and high specificity towards their target. The SPR biosensor had limits of detection of 13.8 and 7.5 nM for Apt-2.5 and Apt-1.4, respectively, which are more than three orders of magnitude lower than the physiological concentrations found in rat urine. Selectivity of the aptamers, when comparing with other major urinary proteins, was excellent, indicating strong efficacy in specific detection of rats. In order to validate the aptamer Apt-2.5 for use with real world samples a FA-based assay was performed on a rat urine sample. The assay showed that the aptamer could detect recombinant MUP13 spiked in filtered urine and the natural MUP13 in unfiltered urine, as a first step into translation to real world application. These are the first known assays to detect and quantify a MUP biomarker of rats.

Novel Electrochemically Switchable, Flexible, Microporous Cloth that Selectively Captures, Releases, and Concentrates Intact Extracellular Vesicles.

There is a significant and growing research interest in the isolation of extracellular vesicles (EVs) from large volumes of biological samples and their subsequent concentration into clean and small volumes of buffers, especially for applications in medical diagnostics. Materials that are easily incorporated into simple sampling devices and which allow the release of EVs without the need for auxiliary and hence contaminating reagents are particularly in demand. Herein, we report on the design and fabrication of a flexible, microporous, electrochemically switchable cloth that addresses the key challenges in diagnostic applications of EVs. We demonstrate the utility of our electrochemically switchable substrate for the fast, selective, nondestructive, and efficient capture and subsequent release of EVs. The substrate consists of an electrospun cloth, infused with a conducting polymer and decorated with gold particles. Utilizing gold-sulfur covalent bonding, the electrospun substrates may be functionalized with SH-terminated aptamer probes selective to EV surface proteins. We demonstrate that EVs derived from primary human dermal fibroblast (HDFa) and breast cancer (MCF-7) cell lines are selectively captured with low nonspecific adsorption using an aptamer specific to the CD63 protein expressed on the EV membranes. The specific aptamer-EV interactions enable easy removal of the nonspecifically bound material through washing steps. The conducting polymer component of the cloth provides a means for efficient (>92%) and fast (<5 min) electrochemical release of clean and intact captured EVs by cathodic cleavage of the Au-S bond. We demonstrate successful capture of diluted EVs from a large volume sample and their release into a small volume of clean phosphate-buffered saline buffer. The developed cloth can easily be incorporated into different designs for separation systems and would be adaptable to other biological entities including cells and other EVs. Furthermore, the capture/release capability holds great promise for liquid biopsies if used to targeted disease-specific markers.