On the Effect of Downscaling in Inkjet Printed Life-Inspired Compartments

The fabrication of size-scalable liquid compartments is a topic of fundamental importance in synthetic biology, aiming to mimic the structures and the functions of cellular compartments. Here, inkjet printing is demonstrated as a customizable approach to fabricate aqueous compartments at different size regimes (from nanoliter to femtoliter scale) revealing the crucial role of size in governing the emerging of new properties. At first, inkjet printing is shown to produce homogenous aqueous compartments stabilized by oil-confinement with mild surfactants down to the hundreds of picoliter scale [1]. Raster Image Correlation Spectroscopy allows to monitor few intermolecular events by the involvement of protein-binding assays within these compartments [2]. Subsequently, in order to reduce droplet size at values below the nozzle size, a theoretical model from Eggers et al. [3] is experimentally reproduced permitting to obtain femtoliter-scale aqueous droplets from picoliter-scale microchannels [4]. As a remarkable difference to picoliter scale droplets, downscaling at the femtoliter-size triggers the spontaneous formation of molecularly crowded shell structures at the water/oil interface stabilized by a mixture of biocompatible surfactants. The shells have typical thickness in order of hundreds of nanometers, in accordance with theoretical models [5]. Molecular crowding effects in these systems are tested by using fluorescence lifetime imaging under the phasor plot approach [6], revealing different characteristic lifetimes of specific probe molecules in the confined volumes with respect to bulk solutions. The femtoliter-scale compartments autonomously trigger the formation of unique features (e.g., up-concentration, spatial heterogeneity, molecular proximity) that are mediated by the intermolecular interactions in these novel environments, ultimately permitting to mimic the native conditions of sub-cellular scale compartments. The crowding conditions in femtoliter-scale droplets do not to affect the conformation variation of a model DNA hairpin in presence of molecular triggers and of a CYP2E1-catalyzed enzymatic reaction. Our results can be a first step towards the fabrication of size-scalable lab-on-a-chip compartments mimicking sub-cellular environments. References 1. G. Arrabito, F. Cavaleri, V. Montalbano, V. Vetri, M. Leone, B. Pignataro, Lab on Chip, 2016, 16, 4666. 2. M.A. Digman, C. M. Brown, A. R. Horwitz,W.W. Mantulin, and E. Gratton, Biophysical Journal, 2016, 94, 2819. 3. J. Eggers, Phys. Rev. Lett. 1993, 71, 3458. 4. G. Arrabito, F. Cavaleri, A. Porchetta, F. Ricci, V. Vetri, M. Leone, B. Pignataro, Adv. Biosys. 2019, 1900023. 5. M. Staszak, J. Surfactants Deterg., 2016, 19, 297. 6. C. Stringari, A. Cinquin, O. Cinquin, M. A. Digman, P.J. Donovan, and E. Gratton, Proc. Natl. Acad. Sci. USA 2011, 108, 13582

Using enhanced number and brightness to measure protein oligomerization dynamics in live cells

Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour

Spectral analysis of pial arteriolar diameter variability during cortical activation in rats suggests a link between vasomotion and neurovascular coupling

L’attività oscillatoria spontanea delle arteriole piali, detta vasomotion, è stata descritta agli inizi degli anni ’80, ma l’impatto dell’attivazione neuronale su tali oscillazioni non è stato analizzato in dettaglio. La stimolazione del nervo sciatico porta ad un incremento del diametro delle arteriole piali sulla corteccia somatosensoriale controlaterale nell’area di rappresentazione dell’arto posteriore. Sfruttando questo ben caratterizzato modello sperimentale per lo studio dell’accoppiamento neurovascolare, il presente studio ha lo scopo di esplorare la vasomotion e le sue modificazioni durante l’accoppiamento neurovascolare mediante un nuovo approccio di analisi. Infatti, per caratterizzare le oscillazioni vascolari, è stata calcolata la potenza totale dello spettro di frequenza nell’intervallo 0.02-2.00 Hz e si è suddiviso questo range in 7 bande sovrapposte del 50%. I risultati indicano che solo le arteriole che si trovano al di sopra della corteccia stimolata mostrano un significativo aumento della potenza totale, a differenza delle arteriole al di sopra della zona di rappresentazione delle vibrisse, usata come controllo per la specificità della risposta vascolare. L’incremento della potenza totale era sostenuto principalmente dall’aumento nel range a bassa frequenza, con due picchi a 0.03 e 0.08 Hz, e da un diffuso aumento nel range frequenziale alto (0.60-2.00 Hz) dello spettro medio. Queste modificazioni dello spettro correlate all’attività suggeriscono: i) la possibilità di valutare le risposte vascolari usando la potenza totale; ii) l’esistenza di almeno tre meccanismi coinvolti nell’accoppiamento neurovascolare: due con una frequenza di feedback nel range a bassa frequenza ed un altro ad alta frequenza; iii) un potenziale coinvolgimento della vasomotion nell’accoppiamento neurovascolare. Inoltre, questi dati mettono in luce la natura oscillatoria dei meccanismi di controllo dell’accoppiamento neurovascolare

Involvement of purinergic nerves in the NANC inhibitory junction potentials in pigeon oesophageal smooth muscle

1. Electrical field stimulation (EFS) (0.5 ms in train of 2-32 Hz for 300 ms) in smooth muscle of pigeon oesophagus, in the presence of atropine (1 microm) and guanethidine (1 microm), elicited an inhibitory response consisting of a transient hyperpolarization (inhibitory junction potential, IJP) associated with muscle relaxation. 2. Sodium nitroprusside (SNP, 100 microm) induced hyperpolarization correlated to mechanical relaxation. 3. The nitric oxide (NO) synthase inhibitor N(omega)-nitro-l-arginine (from 0.1 to 100 microm) caused a concentration-dependent reduction of electromechanical response to EFS indicating a role for NO in this response. 4. Apamin (1 microm) reduced both IJP and relaxation to EFS but was without effect on the response to SNP indicating a role for purines, which are also blocked by apamin. 5. Adenosine, AMP, ADP and ATP (all from 1 microm to 1 mm) application caused transient hyperpolarization and muscular relaxation with the following order of potency: adenosine > AMP > ADP > ATP. 6. Inhibitory responses evoked by purines are TTX (1 microm) insensitive but they were inhibited by apamin. This indicates that a purine component for the non-adrenergic non-cholinergic (NANC) response exists but the purine receptor site is not located on the neurone. 7. Overall these results suggest that NANC inhibitory response elicited by EFS presents two different components apamin-sensitive, probably purines-mediated and apamin-insensitive probably NO-mediated as apamin only partially block the response to EFS