Synthetic antigen-conjugated DNA systems for antibody detection and characterization

Antibodies are among the most relevant biomolecular targetsfordiagnostic and clinical applications. In this Perspective, we providea critical overview of recent research efforts focused on the developmentand characterization of devices, switches, and reactions based onthe use of synthetic antigen-conjugated DNA strands designed to beresponsive to specific antibodies. These systems can find applicationsin sensing, drug-delivery, and antibody-antigen binding characterization.The examples described here demonstrate how the programmability andchemical versatility of synthetic nucleic acids can be used to createinnovative analytical tools and target-responsive systems with promisingpotentials

Simulated microgravity triggers epithelial mesenchymal transition in human keratinocytes

The microgravitational environment is known to affect the cellular behaviour inducing modulation of gene expression and enzymatic activities, epigenetic modifications and alterations of the structural organization. Simulated microgravity, obtained in the laboratory setting through the use of a Random Positioning Machine (RPM), represents a well recognized and useful tool for the experimental studies of the cellular adaptations and molecular changes in response to weightlessness. Short exposure of cultured human keratinocytes to the RPM microgravity influences the cellular circadian clock oscillation. Therefore, here we searched for changes on the regenerative ability and response to tissue damage of human epidermal cells through the analysis of the effects of the simulated microgravity on the re-epithelialization phase of the repair and wound healing process. Combining morphological, biochemical and molecular approaches, we found that the simulated microgravity exposure of human keratinocytes promotes a migratory behavior and triggers the epithelial-mesenchymal transition (EMT) through expression of the typical EMT transcription factors and markers, such as Snail1, Snail2 and ZEB2, metalloproteases, mesenchymal adhesion molecules and cytoskeletal components

Non-natural protein-protein communication mediated by a DNA-based, antibody-responsive device

We report here the rational design and optimization of an antibody responsive, DNA-based device that enables communication between pairs of otherwise non-interacting proteins. The device is designed to recognize and bind a specific antibody and, in response, undergo a conformational change that leads to the release of a DNA strand, termed the “translator,” that regulates the activity of a downstream target protein. As proof of principle, we demonstrate antibody-induced control of the proteins thrombin and Taq DNA polymerase. The resulting strategy is versatile and, in principle, can be easily adapted to control artificial protein-protein communication in artificial regulatory networks

Programmable cell-free transcriptional switches for antibody detection

We report here the development of a cell-free in-vitro transcription system for the detection of specific target antibodies. The approach is based on the use of programmable antigen-conjugated DNA-based conformational switches that, upon binding to a target antibody, can trigger the cell-free transcription of a light-up fluorescence-activating RNA aptamer. The system couples the unique programmability and responsiveness of DNA-based systems with the specificity and sensitivity offered by invitro genetic circuitries and commercially available transcription kits. We demonstrate that cell-free transcriptional switches can efficiently measure antibody levels directly in blood serum. Thanks to the programmable nature of the sensing platform the method can be adapted to different antibodies: we demonstrate here the sensitive, rapid and cost-effective detection of three different antibodies and the possible use of this approach for the simultaneous detection of two antibodies in the same solution

Water Framework Directive Watch List Method Analytical method for determination of compounds selected for the first Surface Water Watch List

Validation of an analytical method is a necessary step in controlling the quality of quantitative analysis. Method validation is an established process which provides documentary evidence that a system fulfils its pre-defined specification, or shows that an analytical method is acceptable for its intended purpose. The purpose of the present study was to develop and validate analytical procedures for the quantitative determination in surface water of substances selected in the First Watch List. Two different methods were developed and validated: • A multi-residual method based on SPE-LC-MS/MS analysis, using OASIS HLB as sorbent material for the extraction of 1 litre water samples and quantitative determination of EE2, E2, E1, diclofenac, azithromycin, clarythromycin, methiocarb acetamiprid, clothianidin, imidacloprid, thiacloprid, thiametoxam and oxadiazon. • A multi-residual method based on LLE-GC-MS, using hexane as extraction solvent for the extraction of 0.01 litre water samples and quantitative determination of BHT, EHMC and Triallate. The calibration curves, working ranges, recoveries, detection and quantification limits, trueness as well as repeatability were determined. The uncertainty budget was estimated based on in-house validation data. JRC.H.1-Water Resource

A multi-sensor approach for volcanic ash cloud retrieval and eruption characterization: the 23 November 2013 Etna lava fountain

Volcanic activity is observed worldwide with a variety of ground and space-based remote sensing instruments, each with advantages and drawbacks. No single system can give a comprehensive description of eruptive activity, and so, a multi-sensor approach is required. This work integrates infrared and microwave volcanic ash retrievals obtained from the geostationary Meteosat Second Generation (MSG)-Spinning Enhanced Visible and Infrared Imager (SEVIRI), the polar-orbiting Aqua-MODIS and ground-based weather radar. The expected outcomes are improvements in satellite volcanic ash cloud retrieval (altitude, mass, aerosol optical depth and effective radius), the generation of new satellite products (ash concentration and particle number density in the thermal infrared) and better characterization of volcanic eruptions (plume altitude, total ash mass erupted and particle number density from thermal infrared to microwave). This approach is the core of the multi-platform volcanic ash cloud estimation procedure being developed within the European FP7-APhoRISM project. The Mt. Etna (Sicily, Italy) volcano lava fountaining event of 23 November 2013 was considered as a test case. The results of the integration show the presence of two volcanic cloud layers at different altitudes. The improvement of the volcanic ash cloud altitude leads to a mean difference between the SEVIRI ash mass estimations, before and after the integration, of about the 30%. Moreover, the percentage of the airborne “fine” ash retrieved from the satellite is estimated to be about 1%–2% of the total ash emitted during the eruption. Finally, all of the estimated parameters (volcanic ash cloud altitude, thickness and total mass) were also validated with ground-based visible camera measurements, HYSPLIT forward trajectories, Infrared Atmospheric Sounding Interferometer (IASI) satellite data and tephra deposits

Single antibody detection in a DNA origami nanoantenna

DNA nanotechnology offers new biosensing approaches by templating different sensor and transducer components. Here, we combine DNA origami nanoantennas with label-free antibody detection by incorporating a nanoswitch in the plasmonic hotspot of the nanoantenna. The nanoswitch contains two antigens that are displaced by antibody binding, thereby eliciting a fluorescent signal. Single-antibody detection is demonstrated with a DNA origami integrated anti-digoxigenin antibody nanoswitch. In combination with the nanoantenna, the signal generated by the antibody is additionally amplified. This allows the detection of single antibodies on a portable smartphone microscope. Overall, fluorescence-enhanced antibody detection in DNA origami nanoantennas shows that fluorescence-enhanced biosensing can be expanded beyond the scope of the nucleic acids realm

Closed-circuit system for the depuration of mussels experimentally contaminated with hepatitis A virus.

In Italy, the consumption of raw or slightly cooked mussels represents the most important risk factor for the transmission of hepatitis A virus (HAV). Although there exist effective methods for the bacterial depuration of contaminated mussels, these methods are poorly effective on enteric viruses. The objective of the present study was to evaluate the effectiveness of a closed-circuit depuration system that uses both ozone and UV light for disinfecting water and that allows salinity and temperature, important parameters for the metabolism of mussels (Mytilus galloprovincialis), to be maintained at constant levels. The results showed that this depuration method decreased the viral load (from 1.72 log 50% tissue culture infective dose [TClD 50 ] ml -1 to <1 log TCID 50 ml -1 within 24 h and from 3.82 log TCID 50 ml -1 to <1 log TCID 50 ml -1 within 48 h). However, in both cases, after 120 h of depuration, a residual amount of virus capable of replicating in cells was detected. These results show that depuration, even if performed with advanced systems, may not guarantee the absence of virus

Microgravity Induces Transient EMT in Human Keratinocytes by Early Down-Regulation of E-Cadherin and Cell-Adhesion Remodeling

Abstract: Changes in cell–matrix and cell-to-cell adhesion patterns are dramatically fostered by the microgravity exposure of living cells. The modification of adhesion properties could promote the emergence of a migrating and invasive phenotype. We previously demonstrated that short exposure to the simulated microgravity of human keratinocytes (HaCaT) promotes an early epithelial– mesenchymal transition (EMT). Herein, we developed this investigation to verify if the cells maintain the acquired invasive phenotype after an extended period of weightlessness exposure. We also evaluated cells’ capability in recovering epithelial characteristics when seeded again into a normal gravitational field after short microgravity exposure. We evaluated the ultra-structural junctional features of HaCaT cells by Transmission Electron Microscopy and the distribution pattern of vinculin and E-cadherin by confocal microscopy, observing a rearrangement in cell–cell and cell–matrix interactions. These results are mirrored by data provided by migration and invasion biological assay. Overall, our studies demonstrate that after extended periods of microgravity, HaCaT cells recover an epithelial phenotype by re-establishing E-cadherin-based junctions and cytoskeleton remodeling, both being instrumental in promoting a mesenchymal–epithelial transition (MET). Those findings suggest that cytoskeletal changes noticed during the first weightlessness period have a transitory character, given that they are later reversed and followed by adaptive modifications through which cells miss the acquired mesenchymal phenotyp