The efficacy of transcranial current stimulation techniques to modulate resting-state EEG, to affect vigilance and to promote sleepiness

Transcranial Current Stimulations (tCSs) are non-invasive brain stimulation techniques which modulate cortical excitability and spontaneous brain activity by the application of weak electric currents through the scalp, in a safe, economic, and well-tolerated manner. The direction of the cortical effects mainly depend on the polarity and the waveform of the applied current. The aim of the present work is to provide a broad overview of recent studies in which tCS has been applied to modulate sleepiness, sleep, and vigilance, evaluating the efficacy of different stimulation techniques and protocols. In recent years, there has been renewed interest in these stimulations and their ability to affect arousal and sleep dynamics. Furthermore, we critically review works that, by means of stimulating sleep/vigilance patterns, in the sense of enhancing or disrupting them, intended to ameliorate several clinical conditions. The examined literature shows the efficacy of tCSs in modulating sleep and arousal pattern, likely acting on the top-down pathway of sleep regulation. Finally, we discuss the potential application in clinical settings of this neuromodulatory technique as a therapeutic tool for pathological conditions characterized by alterations in sleep and arousal domains and for sleep disorders per se

Intelligent Transport Systems: how to Manage a Research in a new Field in IS

This paper sheds light on the management of a research project in a new topic for IS like the one of Intelligent Transport Systems (ITS). It describes and discusses the methodology adopted for a survey designed by the authors and experimented during a recent research on ITS carried out on behalf of an Italian Ministry. The paper presents the first results of this research and draw some conclusions on the problems that have to be faced in order to successfully manage such type of research projects and to build a common knowledge base on ITS.This paper sheds light on the management of a research project in a new topic for IS like the one of Intelligent Transport Systems (ITS). It describes and discusses the methodology adopted for a survey designed by the authors and experimented during a recent research on ITS carried out on behalf of an Italian Ministry. The paper presents the first results of this research and draw some conclusions on the problems that have to be faced in order to successfully manage such type of research projects and to build a common knowledge base on ITS.Monograph's chapter

Supporting Policy Definition in the e-Health domain: a QCA based method

eHealth is broadly considered as a promising strategy to improve the economic sustainability and quality of the healthcare service provision in Europe. Nevertheless, despite the enthusiastic declarations of eHealth potential, the adoption of IT in health care has progressed very slowly. A critical factor, not deeply addressed in literature, is related to the process of prioritization of the eHealth solution to adopt, in presence of financial constrains, external and internal pressure from a wide range of heterogeneous stakeholders, and conflicting information on different technological solutions. In this paper we introduce a method supporting policy definition in the eHealth domain. This method is based on a qualitative comparative analysis (QCA) of best practices and previous experiences performed through the lens of an analytic framework whose dimensions and categories are well situated in the eHealth context. This method could support policy-makers in the identification of the properties and characteristics of innovative projects at European level and to analyze the gap between the international scenario and the local context in order to understand trends and dynamics of development, to evaluate the best opportunities for innovation and, therefore, to assign priorities for the next investments by respecting the constraints of available resources.eHealth is broadly considered as a promising strategy to improve the economic sustainability and quality of the healthcare service provision in Europe. Nevertheless, despite the enthusiastic declarations of eHealth potential, the adoption of IT in health care has progressed very slowly. A critical factor, not deeply addressed in literature, is related to the process of prioritization of the eHealth solution to adopt, in presence of financial constrains, external and internal pressure from a wide range of heterogeneous stakeholders, and conflicting information on different technological solutions. In this paper we introduce a method supporting policy definition in the eHealth domain. This method is based on a qualitative comparative analysis (QCA) of best practices and previous experiences performed through the lens of an analytic framework whose dimensions and categories are well situated in the eHealth context. This method could support policy-makers in the identification of the properties and characteristics of innovative projects at European level and to analyze the gap between the international scenario and the local context in order to understand trends and dynamics of development, to evaluate the best opportunities for innovation and, therefore, to assign priorities for the next investments by respecting the constraints of available resources.Uninvited Submission

Synthesis and Structural Studies on DNA G-Quadruplexes: from Nucleic Acid Aptamers to Higher Order Assemblies.

Quadruple helices, or G-quadruplexes, are DNA secondary structures found in guanine rich oligonucleotide sequences, having a natural propensity to self-associate in coplanar arrays of four guanines, stabilized by Hoogsteen hydrogen bonding. The scientific interest towards these particular DNA structures is mainly due to the presence of guanine rich domains, potentially able to form G-quadruplexes, in important regions of the human genome, as gene promoters and telomeres, and to the fact that the G-quadruplexes can constitute the scaffold of aptamers. Aptamers are short DNA or RNA fragments capable to bind with high affinity specific proteins, as for example thrombin or HIV-proteins. On these grounds, aptamers-based synthetic oligonucleotides can represent a new class of pharmacologically interesting molecules, characterized by a high selectivity of action. Furthermore, the Gquadruplexes can have a potential use in nanotechnology. As a matter of fact, the overall quadruplex scaffold can exhibit several morphologies through intramolecular or intermolecular organization of G-rich oligonucleotide strands, which can form higher-order assemblies by multimerization between G-quadruplex units. In these contests, my research studies have been mainly focused on: i) Investigation of the most prominent biological properties of selected Gquadruplexes and identification of bioactive sequences able to play a biological role as aptamers; and ii) Elucidation of the sequence-specific thermodynamic stability and physicalchemical mechanisms that underlie the G-quadruplexes multimerization. Specifically, during my PhD research studies, I focused on anti-HIV aptamers, and Thrombin Binding Aptamer (TBA) with the aim to identify the structural features required for their biological activity and reaching the way to improve them. Furthermore, I identify the CGGXGGT oligonucleotide sequence (where X = A, C, G, or T) able to lead to the formation of higher order G-quadruplex assemblies, that can potentially be used as DNA-based nanostructures. In order to achieve these objectives, it has been necessary to exploit the synthesis and the structural characterization of quadruplex-forming oligonucleotides, the analysis of the sequence-specific thermodynamic stability, the physical-chemical properties and the structural features of resulting G-quadruplexes. Furthermore, as regard the studies about aptamers, the evaluation of the binding properties to the selected proteins, and bioassays to ascertain the biological properties of the synthesized complexes have been undertaken

Characterization of Glycosylated Proteins at Subunit Level by HILIC/MS

Hydrophilic interaction chromatography (HILIC) coupled to mass spectrometry (MS) is considered as the reference analytical technique for glycans profiling, especially for the characterization of glycosylated protein therapeutics such as monoclonal antibodies (mAbs) and mAbs-related products. Although HILIC/MS is mainly known to profile enzymatically released and fluorescently labeled N-glycans, the recent commercialization of new widepore HILIC amide bonded stationary phases packed with sub-2 μm particles has allowed for remarkable separations also at the subunit level. Here, we describe a simple protocol to perform the mAb glycans profiling at subunit level by HILIC/MS

DNA and RNA telomeric G-quadruplexes: what topology features can be inferred from ion mobility mass spectrometry?

Maintenance of the telomeres is key to chromosome integrity and cell proliferation. The G-quadruplex structures formed by telomeric DNA and RNA (TTAGGG and UUAGGG repeats, respectively) are key to this process. However, because these sequences are particularly polymorphic, solving high-resolution structures is not always possible, and there is a need for new methodologies to characterize the multiple structures coexisting in solution. In this context, we evaluated whether ion mobility spectrometry coupled to native mass spectrometry could help separate and assign the G-quadruplex topologies. We explored the circular dichroism spectra, multimer formation, cation binding, and ion mobility spectra of several 4-repeat and 8-repeat telomeric DNA and RNA sequences, both in NH4+ and in K+. In 1 mM K+ and 100 mM trimethylammonium acetate, all RNAs fold intramolecularly (no multimer). In 8-repeat sequences, the subunits are not independent: in DNA the first subunit disfavors the folding of the second one, whereas in RNA the two subunits fold cooperatively via cation-mediated stacking. Ion mobility spectrometry shows that gas-phase structures keep a memory of – but are not identical to – the solution ones. At the native charge states, the loops can rearrange in a variety of ways (unless they are constrained by pre-formed hydrogen bonds), thereby wrapping the core and masking the strand arrangements. Our study highlights that, to progress towards structural assignment from IM-MS experiments, deeper understanding of the solution-to-gas-phase rearrangement mechanisms is warranted

Comprehensive evaluation of zwitterionic hydrophilic liquid chromatography stationary phases for oligonucleotide characterization

Hydrophilic interaction chromatography (HILIC) has been proposed as a valuable alternative to ion-pairing reversed-phase chromatography (IP-RPLC) for oligonucleotide (ON) analysis. In this context, the potential of seven zwitterionic HILIC columns has been evaluated against amide- and poly-hydroxy fructan-functionalized HILIC columns and a C18 column operated under IP-RPLC mode. Based on the retention characteristics of key small molecule pairs, each zwitterionic HILIC column showed a unique radar-shaped profile, suggesting different selectivities for distinct structural differences. Unmodified DNA and RNA samples were then evaluated, and the columns classified based on their retentivity. Two zwitterionic columns were particularly promising in terms of overall resolution, especially for the largest ONs (&gt; 40-mer). Finally, separations between a chemically modified drug-like ON and its closely related impurities were performed. Although the ZIC-cHILIC column showed similar selectivity values as compared to the reference IP-RPLC technique, all columns demonstrated a general decrease in selectivity due to the minor structural differences present in the highly complex samples. This work highlights the utility of zwitterionic HILIC mode for ON analysis and it reveals the importance of understanding columns characteristics – in terms of retention and selectivity – when selecting a stationary phase for specific ON applications.</p

Bispecific antibody characterization by a combination of intact and site-specific/chain-specific LC/MS techniques

Bispecific antibodies (bsAbs) are considered as an important class of biopharmaceutical drugs, with about 160 products in clinical trials. From an analytical point of view, the correct chain-association is one of the most critical challenge to monitor during bsAbs development and production. In the present study, a full analytical workflow has been developed based on the use of various chromatographic modes: size exclusion chromatography (SEC), ion exchange chromatography (IEX), reversed phase liquid chromatography (RPLC), and hydrophilic interaction chromatography (HILIC), all combined with high resolution mass spectrometry (MS). This analytical strategy was applied to Hemlibra® (emicizumab), which is certainly the most successful commercial bsAb to date. Using this strategy, it was possible to monitor the presence of mispaired bsAb species and detect and identify additional post-translational modifications (PTMs)

A New Practice to Monitor the Fabrication Process of Fab-Targeting Ligands from Bevacizumab by LC-MS: Preparation and Analytical Characterization

The objective of this study was to qualitatively evaluate a Fab-targeting ligand preparation containing free thiol groups in the hinge region by using bevacizumab as a model. The evaluation focused on the purification of fragments through a nonaffinity-based process using a centrifugal ultrafiltration technique and mild reduction conditions for the intact production of F(ab’) fragments with specific inter-heavy-chain disulfide bonds cleavage. Under these conditions, F(ab’) fragments with a defined chemical composition were successfully obtained via proteolytic digestion followed by a controlled reduction reaction process maintaining the integrity of the binding sites. The ultrafiltration purification technique appears to be suitable for the removal of the digestive enzyme but inefficient for the removal of Fc fragments, thus requiring additional processing. A suitable analytical strategy was developed, allowing us to demonstrate the reformation of disulfide bridges between the two reduced cysteines within F(ab’) fragments.</p

A generic workflow for the characterization of therapeutic monoclonal antibodies—application to daratumumab

In the present analytical workflow, chromatographic methods have been developed and hyphenated to mass spectrometry (MS) for the characterization of protein size, charge, hydrophobic, and hydrophilic variants of daratumumab. Multiple critical quality attributes (CQAs) were characterized in forced degraded daratumumab sample, using size exclusion, ion exchange (IEX), and hydrophobic interaction (HIC) chromatography coupled to fluorescence detection for relative quantification and fractionation. Mass assignment was performed by using a fast, non-denaturing and universal size exclusion chromatography (SEC) method prior to native MS analysis of the collected fractions (off-line approach). This allowed the identification of N-terminal lysine clipping, and the extent of glycation and oxidation at intact protein level. Finally, middle-up analysis of daratumumab was performed using reversed phase (RPLC) and hydrophilic interaction (HILIC) chromatography coupled to MS to obtain a comprehensive overview of all PTMs after the forced stressed conditions and a fine characterization of the glycosylation profile. Conveniently, the presented workflow maintains the established golden standard non-denaturing chromatography techniques and additionally introduces a straightforward and automated desalting procedure prior to MS analysis. Therefore, it is expected that the off-line coupling of SEC, IEX, and HIC to SEC-MS has great potential to be implemented in routine characterization of mAbs