A Novel Silicon Platform for Selective Isolation, Quantification, and Molecular Analysis of Small Extracellular Vesicles.

INTRODUCTION: Small extracellular vesicles (sEVs), thanks to their cargo, are involved in cellular communication and play important roles in cell proliferation, growth, differentiation, apoptosis, stemness and embryo development. Their contribution to human pathology has been widely demonstrated and they are emerging as strategic biomarkers of cancer, neurodegenerative and cardiovascular diseases, and as potential targets for therapeutic intervention. However, the use of sEVs for medical applications is still limited due to the selectivity and sensitivity limits of the commonly applied approaches. METHODS: Novel sensing solutions based on nanomaterials are arising as strategic tools able to surpass traditional sensor limits. Among these, Si nanowires (Si NWs), realized with cost-effective industrially compatible metal-assisted chemical etching, are perfect candidates for sEV detection. RESULTS: In this paper, the realization of a selective sensor able to isolate, concentrate and quantify specific vesicle populations, from minimal volumes of biofluid, is presented. In particular, this Si NW platform has a detection limit of about 2×105 sEVs/mL and was tested with follicular fluid and blastocoel samples. Moreover, the possibility to detach the selectively isolated sEVs allowing further analyses with other approaches was demonstrated by SEM analysis and several PCRs performed on the RNA content of the detached sEVs. DISCUSSION: This platform overcomes the limit of detection of traditional methods and, most importantly, preserves the biological content of sEVs, opening the route toward a reliable liquid biopsy analysis.

Modeling, Fabrication and Integration of Wearable Smart Sensors in a Monitoring Platform for Diabetic Patients.

The monitoring of some parameters, such as pressure loads, temperature, and glucose level in sweat on the plantar surface, is one of the most promising approaches for evaluating the health state of the diabetic foot and for preventing the onset of inflammatory events later degenerating in ulcerative lesions. This work presents the results of sensors microfabrication, experimental characterization and FEA-based thermal analysis of a 3D foot-insole model, aimed to advance in the development of a fully custom smart multisensory hardware-software monitoring platform for the diabetic foot. In this system, the simultaneous detection of temperature-, pressure- and sweat-based glucose level by means of full custom microfabricated sensors distributed on eight reading points of a smart insole will be possible, and the unit for data acquisition and wireless transmission will be fully integrated into the platform. Finite element analysis simulations, based on an accurate bioheat transfer model of the metabolic response of the foot tissue, demonstrated that subcutaneous inflamed lesions located up to the muscle layer, and ischemic damage located not below the reticular/fat layer, can be successfully detected. The microfabrication processes and preliminary results of functional characterization of flexible piezoelectric pressure sensors and glucose sensors are presented. Full custom pressure sensors generate an electric charge in the range 0-20 pC, proportional to the applied load in the range 0-4 N, with a figure of merit of 4.7 ± 1 GPa. The disposable glucose sensors exhibit a 0-6 mM (0-108 mg/dL) glucose concentration optimized linear response (for sweat-sensing), with a LOD of 3.27 µM (0.058 mg/dL) and a sensitivity of 21 µA/mM cm2 in the PBS solution. The technical prerequisites and experimental sensing performances were assessed, as preliminary step before future integration into a second prototype, based on a full custom smart insole with enhanced sensing functionalities.

PDMS/Kapton interface plasma treatment effects on the polymeric package for a wearable thermoelectric generator.

The present work highlights the progress in the field of polymeric package reliability engineering for a flexible thermoelectric generator realized by thin-film technology on a Kapton substrate. The effects of different plasma treatments on the mechanical performance at the interface of a poly(dimethylsiloxane) (PDMS)/Kapton assembly were investigated. To increase the package mechanical stability of the realized wearable power source, the Kapton surface wettability after plasma exposure was investigated by static contact-angle measurements using deionized water and PDMS as test liquids. In fact, the well-known weak adhesion between PDMS and Kapton can lead to a delamination of the package with an unrecoverable damage of the generator. The plasma effect on the adhesion performances was evaluated by the scratch-test method. The best result was obtained by performing a nitrogen plasma treatment at a radio-frequency power of 20 W and a gas flow of 20 sccm, with a measured critical load of 1.45 N, which is 2.6 times greater than the value measured on an untreated Kapton substrate and 1.9 times greater than the one measured using a commercial primer.

RNA polymerase III transcription - regulated by chromatin structure and regulator of nuclear chromatin organization.

RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its transcription factors have been implicated in the organization of nuclear chromatin in several organisms. In yeast, the ability of the Pol III transcription system to contribute to nuclear organization seems to be dependent on direct interactions of Pol III genes and/or its transcription factors TFIIIC and TFIIIB with the structural maintenance of chromatin (SMC) protein-containing complexes cohesin and condensin. In human cells, Pol III genes and transcription factors have also been shown to colocalize with cohesin and the transcription regulator and genome organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been identified in yeast and humans that are bound by partial Pol III machineries (extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC as well as Pol III genes possess the ability to act as boundary elements that restrict spreading of heterochromatin.

RNA polymerase III transcription control elements: themes and variations.

Eukaryotic genomes are punctuated by a multitude of tiny genetic elements, that share the property of being recognized and transcribed by the RNA polymerase (Pol) III machinery to produce a variety of small, abundant non-protein-coding (nc) RNAs (tRNAs, 5S rRNA, U6 snRNA and many others). The highly selective, efficient and localized action of Pol III at its minute genomic targets is made possible by a handful of cis-acting regulatory elements, located within the transcribed region (where they are bound by the multisubunit assembly factor TFIIIC) and/or upstream of the transcription start site. Most of them participate directly or indirectly in the ultimate recruitment of TFIIIB, a key multiprotein initiation factor able to direct, once assembled, multiple transcription cycles by Pol III. But the peculiar efficiency and selectivity of Pol III transcription also depends on its ability to recognize very simple and precisely positioned termination signals. Studies in the last few years have significantly expanded the set of known Pol III-associated loci in genomes and, concomitantly, have revealed unexpected features of Pol III cis-regulatory elements in terms of variety, function, genomic location and potential contribution to transcriptome complexity. Here we review, in a historical perspective, well established and newly acquired knowledge about Pol III transcription control elements, with the aim of providing a useful reference for future studies of the Pol III system, which we anticipate will be numerous and intriguing for years to come.

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III.

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3'-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T(≥4) stretch within 50 bp of 3'-flanking region. In vitro analysis of tDNAs with a distanced T(≥4) revealed the existence of non-canonical terminators resembling degenerate T(≥5) elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3' trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3'-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3'-trailer sequences with the potential to contribute novel functional ncRNAs.

The telomere-binding protein Tbf1 demarcates snoRNA gene promoters in Saccharomyces cerevisiae.

Small nucleolar RNAs (snoRNAs) play a key role in ribosomal RNA biogenesis, yet factors controlling their expression are unknown. We found that the majority of Saccharomyces snoRNA promoters display an aRCCCTaa sequence motif at the upstream border of a TATA-containing nucleosome-free region. Genome-wide ChIP-seq analysis showed that these motifs are bound by Tbf1, a telomere-binding protein known to recognize mammalian-like T(2)AG(3) repeats at subtelomeric regions. Tbf1 has over 100 additional promoter targets, including several other genes involved in ribosome biogenesis and the TBF1 gene itself. Tbf1 is required for full snoRNA expression, yet it does not influence nucleosome positioning at snoRNA promoters. In contrast, Tbf1 contributes to nucleosome exclusion at non-snoRNA promoters, where it selectively colocalizes with the Tbf1-interacting zinc-finger proteins Vid22 and Ygr071c. Our data show that, besides the ribosomal protein gene regulator Rap1, a second telomere-binding protein also functions as a transcriptional regulator linked to yeast ribosome biogenesis.

General transcription factors and subunits of RNA polymerase III: Paralogs for promoter- and cell type-specific transcription in multicellular eukaryotes.

In the course of evolution of multi-cellular eukaryotes, paralogs of general transcription factors and RNA polymerase subunits emerged. Paralogs of transcription factors and of the RPC32 subunit of RNA polymerase III play important roles in cell type- and promoter-specific transcription. Here we discuss their respective functions.