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Triple helix DNA has been exploited in building molecular switches, based on Watson–Crick and Hoogsteen base pairings, and adapted to several biosensing strategies for the detection of nucleic acids and aptasensors development [1-4]. The stability of the triple helix stem can be higher compared to longer duplex, and good sensitivity and selectivity have been generally observed in sensing. Moreover, different recognition elements specific for different targets can be included in the probe on a same single-stranded sequence, allowing in principle a universal assay strategy [5]. Examples in literature in using triple helix DNA probes usually employ only one of the involved oligonucleotides for signal amplification and transduction. We herein propose the use of both the triple helix forming strands in this process as this would give an advantage, such as the chance to combine different transduction approaches leading to a stronger and more reliable detection method. For this reason, we designed triple helix DNA probes specific for different microRNAs, in which both the sequences hidden in the stem would be available for subsequent signal amplification and transduction after target recognition. (Fig.1). To simply test the capacity of our system to induce a double response in the presence of the target, we designed hybridization chain reactions (HCR) [6] triggered by both the oligonucleotides involved in the triple helix. Furthermore, in our peculiar design, the self-assembled nanostructures resulting from a single recognition event can interact with each other forming bigger nanostructures, thus enhancing the recognition signal. After characterizing this strategy in solution (see Fig. 2), we are now in the process of implementing it in a biosensor platform.

The triple helix is an alternative structure adopted by nucleic acids based on Watson–Crick and Hoogsteen base pairings involving three strands in the correct orientation. Since the stability of the triple helix stem can be higher even compared to longer duplexes, triple stranded DNA has been used in biosensing to build aptasensors or sensors for nucleic acids detection [1-4] and good sensitivity and selectivity have been generally observed. Moreover, different recognition elements specific for different targets can be included in the nucleic acid probe on a same single-stranded sequence, allowing in principle a universal assay strategy [5]. In the literature, the use of only one of the involved oligonucleotides for signal amplification and transduction is generally reported. We herein propose the use of both the triple helix forming strands in this process as this would give an advantage, such as the chance to combine different transduction approaches leading to a stronger and more reliable detection method. For this reason, we designed triple helix DNA probes specific for different microRNAs, in which both the sequences hidden in the stem would be available for subsequent signal amplification and transduction after target recognition (Fig. 1). To simply test the feasibility of our system to induce a double response triggered by the target, we designed hybridization chain reactions (HCR) [6] started by both the oligonucleotides involved in the triple helix. Furthermore, in our peculiar design, the self-assembled nanostructures resulting from a single recognition event can interact with each other forming bigger nanostructures, thus enhancing the recognition signal. After characterizing this strategy in solution (see Fig. 2), we are now in the process of implementing it in a biosensor platform.

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Journal of Medicinal Chemistry, 61, 7640–7656 (DOI: 10.1021/acs.jmedchem.8b00610), © 2018 American Chemical Society, after peer review and technical editing by the publisher. To access the final edited and published work, see https://pubs.acs.org/articlesonrequest/AOR-yrCZV6e6VNr5AFhKcH79 This Manuscript version is made available under the CC-BY-NC-ND 4.0 license. https://creativecommons.org/licenses/by-nc-nd/4.0/ ABSTRACT Several findings propose altered tau protein network as an important target for Alzheimer’s disease (AD). Particularly, two points of pharmacological intervention can be envisaged: inhibition of phosphorylating tau kinase GSK-3β, and tau aggregation process. In light of this and based on our interest in multi-target paradigms in AD, we report on the discovery of 2,4-thiazolidinedione-derivatives endowed with such profile. 28 and 30 displayed micromolar IC50 values towards GSK-3β, together with the capacity of inhibiting AcPHF6 aggregation of 60% and 80% at 10 μM, respectively. In addition, they showed PAMPA-BBB permeability, together with a suitable cellular safety profile. 30 also displayed inhibition of both K18 and full-length tau aggregations. Finally, both compounds were able to improve cell viability in an okadaic acid-induced neurodegeneration cell model. To the best of our knowledge, 28 and 30 are the first balanced, non-toxic, dual-acting compounds hitting tau cascade at two different hubs.

Several findings propose the altered tau protein network as an important target for Alzheimer's disease (AD). Particularly, two points of pharmacological intervention can be envisaged: inhibition of phosphorylating tau kinase GSK-3β and tau aggregation process. On the basis of this consideration and on our interest in multitarget paradigms in AD, we report on the discovery of 2,4-thiazolidinedione derivatives endowed with such a profile. 28 and 30 displayed micromolar IC values toward GSK-3β, together with the capacity of inhibiting AcPHF6 aggregation of 60% and 80% at 10 μM, respectively. In addition, they showed PAMPA-BBB permeability, together with a suitable cellular safety profile. 30 also displayed inhibition of both K18 and full-length tau aggregations. Finally, both compounds were able to improve cell viability in an okadaic acid-induced neurodegeneration cell model. To the best of our knowledge, 28 and 30 are the first balanced, nontoxic, dual-acting compounds hitting tau cascade at two different hubs., This work was supported by the University of Bologna (RFO2015_2016), European Cooperation of Science and Technology (EUCOST) Action CA15135, Ministerio de Economia y Competitividad (CTQ2015-66313-R), Italian Ministry of Health (GR-2013-02355724), MJFF, ALZ, Alzheimer’s Research UK and the Weston Brain Institute (BAND2015). The authors thank E. De Cecco (PhD candidate, SISSA) for providing K18 and 2N4R protein samples for the in vitro assays.

We investigated the dewetting process on flat and chemically patterned surfaces of ultrathin films (thickness between 2 and 15 nm) of a cylinder forming polystyrene- block-poly(methyl methacrylate) (PS- b-PMMA) spin coated on poly(styrene- r-methyl methacrylate) random copolymers (RCPs). When the PS- b-PMMA film dewets on a 2 nm-thick RCP layer, the ordering of the hexagonally packed PMMA cylinders in the dewetted structures extends over distances far exceeding the correlation length obtained in continuous block copolymer (BCP) films. As a result, micrometer-sized circular droplets featuring defectless single grains of self-assembled PS- b-PMMA with PMMA cylinders perpendicularly oriented with respect to the substrate are generated and randomly distributed on the substrate. Additionally, alignment of the droplets along micrometric lines was achieved by performing the dewetting process on large-scale chemically patterned stripes of 2 nm thick RCP films by laser lithography. By properly adjusting the periodicity of the chemical pattern, it was possible to tune and select the geometrical characteristics of the dewetted droplets in terms of maximum thickness, contact angle and diameter while maintaining the defectless single grain perpendicular cylinder morphology of the circular droplets.

DNA methylation catalyzed by DNA methyl transferase (MTase) is a significant epigenetic process for modulating gene expression. Abnormal levels of DNA MTase enzyme have been regarded as a cancer biomarker or a sign of bacterial diseases. We developed a novel colorimetric method to assay M.SssI MTase activity employing peroxidase-like activity of DNA template Ag/Pt NCs without using restriction enzymes. Based on inhibiting the peroxidase reaction that occurred in the TMB-H2O2 system, in the presence of MTase, a highly sensitive and selective colorimetric biosensor was fabricated with a detection limit (LOD) of 0.05 U/mL and a linear range from 0.5 to 10 U/mL. The changes in absorption intensity were monitored to quantify the M.SssI activity. This strategy had a high selectivity over other proteins. Furthermore, it is also demonstrated that this method can be used for the evaluation and screening of inhibitors for DNA MTase.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Mutations in the gene encoding copper/zinc superoxide dismutase-1 (SOD1) are responsible for most familiar cases, but the role of mutant SOD1 protein dysfunction in non-cell autonomous neurodegeneration, especially in relation to microglial activation, is still unclear. Here, we focused our study on microglial cells, which release SOD1 also through exosomes. We observed that in rat primary microglia the overexpression of the most-common SOD1 mutations linked to fALS (G93A and A4V) leads to SOD1 intracellular accumulation, which correlates to autophagy dysfunction and microglial activation. In primary contact co-cultures, fALS mutant SOD1 overexpression by microglial cells appears to be neurotoxic by itself. Treatment with the autophagy-inducer trehalose reduced mutant SOD1 accumulation in microglial cells, decreased microglial activation and abrogated neurotoxicity in the co-culture model. These data suggest that i) the alteration of the autophagic pathway due to mutant SOD1 overexpression is involved in microglial activation and neurotoxicity; ii) the induction of autophagy with trehalose reduces microglial SOD1 accumulation through proteasome degradation and activation, leading to neuroprotection. Our results provide a novel contribution towards better understanding key cellular mechanisms in non-cell autonomous ALS neurodegeneration.

DNA biosensors could overcome some of the common drawbacks of lab-based techniques for nucleic acids detection for diagnostics purposes. One of the main impediments for such applications of DNA biosensors is their lack of sensitivity: this can prevent their full exploitation in the diagnostic analytical field. DNA nanotechnology could enhance DNA biosensors and let them perform at the required high sensitivity. Well-designed, programmable self-assembly reactions can be triggered by a specific nucleic acid target. The Hybridization Chain Reaction (HCR) is a self-assembly strategy in which the target nucleic acid sequence triggers the formation of long nicked double-stranded DNA nanostructures. This can be performed in solution or on a surface, and the process can be coupled to different signal transduction schemes. We here describe the methods to design and test HCR reactions for the detection of different nucleic acid targets in solution and the procedures to exploit this strategy on surfaces with an electrochemical biosensing platform.