A Multi-Layered Study on Harmonic Oscillations in Mammalian Genomics and Proteomics

Cellular, organ, and whole animal physiology show temporal variation predominantly featuring 24-h (circadian) periodicity. Time-course mRNA gene expression profiling in mouse liver showed two subsets of genes oscillating at the second (12-h) and third (8-h) harmonic of the prime (24-h) frequency. The aim of our study was to identify specific genomic, proteomic, and functional properties of ultradian and circadian subsets. We found hallmarks of the three oscillating gene subsets, including different (i) functional annotation, (ii) proteomic and electrochemical features, and (iii) transcription factor binding motifs in upstream regions of 8-h and 12-h oscillating genes that seemingly allow the link of the ultradian gene sets to a known circadian network. Our multifaceted bioinformatics analysis of circadian and ultradian genes suggests that the different rhythmicity of gene expression impacts physiological outcomes and may be related to transcriptional, translational and post-translational dynamics, as well as to phylogenetic and evolutionary components

Assembling patchy plasmonic nanoparticles with aggregation-dependent antibacterial activity

We realise an antibacterial nanomaterial based on the self-limited assembly of patchy plasmonic colloids, obtained by adsorption of lysozyme to gold nanoparticles. The possibility of selecting the size of the assemblies within several hundred nanometres allows for tuning their optical response in a wide range of frequencies from visible to near infrared. We also demonstrate an aggregation-dependent modulation of the catalytic activity, which results in an enhancement of the antibacterial performances for assemblies of the proper size. The gained overall control on structure, optical properties and biological activity of such nanomaterial paves the way for the development of novel antibacterial nanozymes with promising applications in treating multi drug resistant bacteria

Toward a unified description of the electrostatic assembly of microgels and nanoparticles

The combination of soft responsive particles, such as microgels, with nanoparticles (NPs) yields highly versatile complexes of great potential for applications, from ad-hoc plasmonic sensors to controlled protocols for loading and release. However, the assembly process between these microscale networks and the co-dispersed nano-objects has not been investigated so far at the microscopic level, preempting the possibility of designing such hybrid complexes a priori. In this work, we combine state-of-the-art numerical simulations with experiments, to elucidate the fundamental mechanisms taking place when microgels-NPs assembly is controlled by electrostatic interactions. We find a general behavior where, by increasing the number of interacting NPs, the microgel deswells up to a minimum size, after which a plateau behavior occurs. This occurs either when NPs are mainly adsorbed to the microgel corona via the folding of the more external chains, or when NPs penetrate inside the microgel, thereby inducing a collective reorganization of the polymer network. By varying microgel properties, such as fraction of crosslinkers or charge, as well as NPs size and charge, we further show that the microgel deswelling curves can be rescaled onto a single master curve, for both experiments and simulations, demonstrating that the process is entirely controlled by the charge of the whole microgel-NPs complex. Our results thus have a direct relevance in fundamental materials science and offer novel tools to tailor the nanofabrication of hybrid devices of technological interest

Molecular origin of the two-step mechanism of gellan aggregation

Among hydrocolloids, gellan is one of the most studied polysaccharides due to its ability to form mechanically stable gels. Despite its long-standing use, the gellan aggregation mechanism is still not understood because of the lack of atomistic information. Here, we fill this gap by developing a new gellan force field. Our simulations offer the first microscopic overview of gellan aggregation, detecting the coil to single-helix transition at dilute conditions and the formation of higher-order aggregates at high concentration through a two-step process: first, the formation of double helices and then their assembly into superstructures. For both steps, we also assess the role of monovalent and divalent cations, complementing simulations with rheology and atomic force microscopy experiments and highlighting the leading role of divalent cations. These results pave the way for future use of gellan-based systems in a variety of applications, from food science to art restoration

The Complex Story Behind a Deep Eutectic Solvent Formation as Revealed by L‑Menthol Mixtures with Butylated Hydroxytoluene Derivatives.

An in-depth study of the hydrophobic eutectic mixtures formed by L-menthol (MEN) with the butylated hydroxytoluene (BHT), 2-tert-butyl-pcresol (TBC), and p-cresol (PC) compounds has been carried out, where TBC and PC are analogous to the BHT species but with a different degree of steric hindrance around the hydroxyl group. Thermal characterization evidenced that the BHT/MEN system can be classified as an ideal eutectic, while the TBC/MEN and PC/MEN mixtures behave as type V deep eutectic solvents (DESs) for a wide range of compositions around the eutectic point. As shown by an array of experimental and theoretical methods, in the BHT/MEN mixtures the establishment of hydrogen-bond (H-bond) interactions between the components is dramatically hampered because of the steric hindrance in the BHT molecule, so that the achievement of a liquid phase at room temperature for the eutectic composition is driven by apolar−apolar attractions among the alkyl functional groups of the constituents. Differently, the TBC-MEN donor−receptor H-bond is the main driving force for the formation of a type V DES and derives from a concurrence of electronic and steric factors characterizing the TBC molecule. Finally, the absence of steric hindrance around the hydroxyl group allows the self-association among PC molecules through H-bonded networks already in the pristine compound, but the replacement with the more favorable PC-MEN H-bond provides a type V DES upon mixing of these components. Our combined approach, together with the peculiarity of the inspected systems, delivered an archetypal study able to shed light onto the various contributions ruling the structure− properties relationship in DESs and possibly deepening the currently accepted view of these inherently complex media

Systematic Analysis of Mouse Genome Reveals Distinct Evolutionary and Functional Properties Among Circadian and Ultradian Genes

In living organisms, biological clocks regulate 24 h (circadian) molecular, physiological, and behavioral rhythms to maintain homeostasis and synchrony with predictable environmental changes, in particular with those induced by Earth's rotation on its axis. Harmonics of these circadian rhythms having periods of 8 and 12 h (ultradian) have been documented in several species. In mouse liver, harmonics of the 24-h period of gene transcription hallmarked genes oscillating with a frequency two or three times faster than circadian periodicity. Many of these harmonic transcripts enriched pathways regulating responses to environmental stress and coinciding preferentially with subjective dawn and dusk. At this time, the evolutionary history of genes with rhythmic expression is still poorly known and the role of length-of-day changes due to Earth's rotation speed decrease over the last four billion years is totally ignored. We hypothesized that ultradian and stress anticipatory genes would be more evolutionarily conserved than circadian genes and background non-oscillating genes. To investigate this issue, we performed broad computational analyses of genes/proteins oscillating at different frequency ranges across several species and showed that ultradian genes/proteins, especially those oscillating with a 12-h periodicity, are more likely to be of ancient origin and essential in mice. In summary, our results show that genes with ultradian transcriptional patterns are more likely to be phylogenetically conserved and associated with the primeval and inevitable dawn/dusk transitions

Hybrid plasmonic nanoparticle assemblies with tunable properties for biophysical applications

In recent years nanoplasmonics has attracted increasing scientific interest arising from the possibility of manipulating the optical phenomena at the interface of nanostructured materials. Indeed, advances in nanofabrication techniques enabled for tailoring and enhancing electromagnetic fields at the sub-wavelength scale, opening to a wide range of applications in different scientific contexts, spreading from electronics to biomedicine. Among the different nanoarchitectures available, a relevant position is occupied by systems made of nanoparticles (NPs) of noble metals such as gold and silver. This Ph.D. Thesis is aimed at addressing the design of plasmonic nanostructures with the desired optical and biological properties. The general idea is to reach a strict control on the spatial organisation and surface properties of gold NP assemblies for mastering the plasmon coupling and the interaction with the external environment. Efforts have been made in quantitatively framing the presented studies in the context of nanoplasmonics theory, and in developing interpretative models for the specific phenomena studied. The results were supported by biological investigation with the purpose of providing a strong scientific background in transferring the obtained findings towards the development of novel biophysical strategies

Thermophilic rearrangement of bio-plasmonic aggregates: morphological and plasmonic related evidences

The peculiar interaction of metallic nanoparticles with the electromagnetic radiation paved the way to design novel nanoarchitectures whose optical properties can be tuned by controlling their structure and the features of the surrounding environment. The research of the last few years heads up to the idea of creating hybrid assemblies made up of metallic nanoparticles and biomolecules with promising applications in the field of nano-medicine and nano-biotechnology, providing a new and powerful tool for innovative diagnosis and therapeutical approaches. We recently developed a bio-plasmonic system based on the colloidal aggregation in solution of anionic gold nanoparticles (AuNPs) mediated by lysozyme. The aggregation is driven by patch-charge interactions [6], induced by the adsorption of the positively charged protein on the AuNPs surface. We demonstrated that the optical properties of the system can be tuned through the clusters morphology, acting on several parameters such as the AuNPs size, the Lysozyme-AuNPs relative molar ratio and the pH of the solution. Proceeding from these, here we would consider also the role of the temperature as a further tool to fine tuning the structural morphology together with the plasmonic properties of the aggregates. In this framework, the thermally enhanced diffusion of the NPs within the clusters can affect aggregate stability and shape, and thereby the own plasmonic profiles. On the other hand, the unfolding of the protein, induced by the increasing temperature and its consequent relaxation on the AuNps surface [7], implies a redistribution of the surface charge, together with an increase of the hydrophobic interactions. Lysozyme unfolding can thus be employed to change the nature of the interaction which holds the aggregates, switching from electrostatic to hydrophobic. As a first step in this direction we undertook a combined study of the temperature effects on the localized surface plasmon resonance and on the size of preformed Lysozyme-NPs aggregates. The plasmonic profile and the related inter-particles plasmonic bands which arise due to the NPs aggregation were monitored by UV-Visible Absorption Spectroscopy at varying the temperature from 20°C to 90°C, while information on the aggregates size has been obtained by Dynamic Light Scattering experiments. The combination of these techniques allowed us to disentangle the two abovementioned aspects, which can interplay in the stability of the clusters, leading to their disaggregation or resulting in the cluster reorganization, depending on the Lysozyme-AuNPs relative molar ratio. It is well known that the localised heating can be also induced by the plasmonic absorption, hence our work sets the foundations to realize a “thermo-plasmonic based annealing”

SERS active pH-nanosensor with tunable properties

The extraordinary optical properties of gold nanoparticles (AuNPs), together with their rewarding chemical stability and ease of functionalization, make them an invaluable platform to develop ultrasensitive and molecular specific chemical sensors. The huge amplification of the spectroscopic signal of molecules located at the metal interface, arising from the confinement of strong electromagnetic fields on the AuNPs surface, results in a remarkable increase of the sensitivity of vibrational spectroscopies. In particular, Surface Enhanced Raman Spectroscopy (SERS) emerged as a powerful analytical tool with detection limits lowered down to the single molecule recognition. In this framework, we developed and characterized a plasmonic pH-nanosensor by conjugating AuNPs with the pH-sensitive molecular probe 4-mercaptobenzoic acid (4MBA), which shows a SERS signal depending on its protonation degree. pH is indeed a key target parameter in a wide field of applications, ranging from environmental science to industry to biomedicine. Exposing the AuNP-4MBA nanosensors to solutions at varying pH, we identified the dynamic range of sensitivity as a function of the relative intensity of selected pHdependent SERS bands. From the comparison of the obtained calibration curve with a standard acid-base titration curve of the free molecule, we enlightened that the pKa of the molecule shifts to higher values when it is measured by SERS at the interface of the plasmonic nanostructure. In particular, for AuNPs with a diameter of 60 nm, the pKa value results around pH 6, making this system suitable for pH measurements in physiological environment, at the single cell level. Proceeding from this, we will explore the possibility of tuning the 4MBA acidic properties by varying the AuNPs size as well as the core material, with the final aim toreach a modulation of the working point of the nanosensor, depending on the system of interest

Exploiting SERS sensitivity to monitor DNA aggregation properties

In the last decades. DNA has been considered far more than the system carrying the essential genetic instructions. Indeed, because of the remarkable properties of the base-pairing specificity and thermoreversibility of the interactions, DNA plays a central role in the design of innovative architectures at the nanoscale. Here, combining complementary DNA strands with a custom-made solution of silver nanoparticles, we realize plasmonic aggregates to exploit the sensitivity of Surface Enhanced Raman Spectroscopy (SERS) for the identification/detection of the distinctive features of DNA hybridization, both in solution and on dried samples. Moreover. SERS allows monitoring the DNA aggregation process by following the temperature variation of a specific spectroscopic marker associated with the Watson-Crick hydrogen bond formation. This temperature-dependent behavior enables us to precisely reconstruct the melting profile of the selected DNA sequences by spectroscopic measurements only. (C) 2020 Elsevier B.V. All rights reserved