Water slowing down drives the occurrence of the low temperature dynamical transition in microgels.

The protein dynamical transition marks an increase in atomic mobility and the onset of anharmonic motions at a critical temperature (T d), which is considered relevant for protein functionality. This phenomenon is ubiquitous, regardless of protein composition, structure and biological function and typically occurs at large protein content, to avoid water crystallization. Recently, a dynamical transition has also been reported in non-biological macromolecules, such as poly(N-isopropyl acrylamide) (PNIPAM) microgels, bearing many similarities to proteins. While the generality of this phenomenon is well-established, the role of water in the transition remains a subject of debate. In this study, we use atomistic molecular dynamics (MD) simulations and elastic incoherent neutron scattering (EINS) experiments with selective deuteration to investigate the microscopic origin of the dynamical transition and distinguish water and PNIPAM roles. While a standard analysis of EINS experiments would suggest that the dynamical transition occurs in PNIPAM and water at a similar temperature, simulations reveal a different perspective, also qualitatively supported by experiments. From room temperature down to about 180 K, PNIPAM exhibits only modest changes of dynamics, while water, being mainly hydration water under the probed extreme confinement, significantly slows down and undergoes a mode-coupling transition from diffusive to activated. Our findings therefore challenge the traditional view of the dynamical transition, demonstrating that it occurs in proximity of the water mode-coupling transition, shedding light on the intricate interplay between polymer and water dynamics.

Dynamic Personality of Proteins and Effect of the Molecular Environment.

Protein dynamics display distinct traits that are linked to their specific biological function. However, the interplay between intrinsic dynamics and the molecular environment on protein stability remains poorly understood. In this study, we investigate, by incoherent neutron scattering, the subnanosecond time scale dynamics of three model proteins: the mesophilic lysozyme, the thermophilic thermolysin, and the intrinsically disordered ß-casein. Moreover, we address the influence of water, glycerol, and glucose, which create progressively more viscous matrices around the protein surface. By comparing the protein thermal fluctuations, we find that the internal dynamics of thermolysin are less affected by the environment compared to lysozyme and ß-casein. We ascribe this behavior to the protein dynamic personality, i.e., to the stiffer dynamics of the thermophilic protein that contrasts the influence of the environment. Remarkably, lysozyme and thermolysin in all molecular environments reach a critical common flexibility when approaching the calorimetric melting temperature.

Nanoclay-Doped Electrospun Nanofibers for Tissue Engineering: Investigation on the Structural Modifications in Physiological Environment.

Background: Clay minerals are nanomaterials that have recently been recognized as enabling excipients that can promote cell adhesion, proliferation, and differentiation. When nanoclays are loaded in a 3D polymeric nanostructure, the cell-substrate interaction is enhanced, and other bioactive properties are optimized. Purpose: In this study, hectorite (HEC)- and montmorillonite (MMT)-doped polymeric scaffolds were explored for the treatment of deep and chronic skin lesions. Methods: Scaffolds were manufactured by means of electrospinning and then crosslinked by heating. Physicochemical analyses were correlated with in vitro biopharmaceutical characterization to predict the in vivo fate of the clay-doped scaffolds. Results and Discussion: The addition of MMT or HEC to the polymeric scaffold framework modifies the surface arrangement and, consequently, the potential of the scaffolds to interact with biological proteins. The presence of nanoclays alters the nanofiber morphology and size, and MMT doping increases wettability and protein adhesion. This has an impact on fibroblast behavior in a shorter time since scaffold stiffness facilitates cell adhesion and cell proliferation. Conclusion: MMT proved to perform better than HEC, and this could be related to its higher hydrophilicity and protein adhesion.

Microscopic versus Macroscopic Glass Transitions and Relevant Length Scales in Mixtures of Industrial Interest.

We have combined X-ray diffraction, neutron diffraction with polarization analysis, small-angle neutron scattering (SANS), neutron elastic fixed window scans (EFWS), and differential scanning calorimetry (DSC) to investigate polymeric blends of industrial interest composed by isotopically labeled styrene-butadiene rubber (SBR) and polystyrene (PS) oligomers of size smaller than the Kuhn length. The EFWS are sensitive to the onset of liquid-like motions across the calorimetric glass transition, allowing the selective determination of the "microscopic" effective glass transitions of the components. These are compared with the "macroscopic" counterparts disentangled by the analysis of the DSC results in terms of a model based on the effects of thermally driven concentration fluctuations and self-concentration. At the microscopic level, the mixtures are dynamically heterogeneous for blends with intermediate concentrations or rich in PS, while the sample with highest content of the fast SBR component looks as dynamically homogeneous. Moreover, the combination of SANS and DSC has allowed determining the relevant length scale for the α-relaxation through its loss of equilibrium to be ≈30 Å. This is compared with the different characteristic length scales that can be identified in these complex mixtures from structural, thermodynamical, and dynamical points of view because of the combined approach followed. We also discuss the sources of the non-Gaussian effects observed for the atomic displacements and the applicability of a Lindemann-like criterion in these materials.

Polymorphism and Ligand Binding Modulate Fast Dynamics of Human Telomeric G-Quadruplexes.

Telomeric G-quadruplexes (G4s) are promising targets in the design and development of anticancer drugs. Their actual topology depends on several factors, resulting in structural polymorphism. In this study, we investigate how the fast dynamics of the telomeric sequence AG3(TTAG3)3 (Tel22) depends on the conformation. By using Fourier transform Infrared spectroscopy, we show that, in the hydrated powder state, Tel22 adopts parallel and mixed antiparallel/parallel topologies in the presence of K+ and Na+ ions, respectively. These conformational differences are reflected in the reduced mobility of Tel22 in Na+ environment in the sub-nanosecond timescale, as probed by elastic incoherent neutron scattering. These findings are consistent with the G4 antiparallel conformation being more stable than the parallel one, possibly due to the presence of ordered hydration water networks. In addition, we study the effect of Tel22 complexation with BRACO19 ligand. Despite the quite similar conformation in the complexed and uncomplexed state, the fast dynamics of Tel22-BRACO19 is enhanced compared to that of Tel22 alone, independently of the ions. We ascribe this effect to the preferential binding of water molecules to Tel22 against the ligand. The present results suggest that the effect of polymorphism and complexation on the G4 fast dynamics is mediated by hydration water.

Symptom control and health-related quality of life in allergic rhinitis with and without comorbid asthma: A multicentre European study.

BACKGROUND: Allergic rhinitis (AR) is a major non-communicable disease that affects the health-related quality of life (HRQoL) of patients. However, data on HRQoL and symptom control in AR patients with comorbid asthma (AR + asthma) are lacking. METHODS: In this multicentre, cross-sectional study, patients with AR were screened and administered questionnaires of demographic characteristics and health conditions (symptoms/diagnosis of AR and asthma, disease severity level, and allergic conditions). HRQoL was assessed using a modified version of the RHINASTHMA questionnaire (30, 'not at all bothered' - 150 'very much bothered') and symptom control was evaluated by a modified version of the Control of Allergic Rhinitis/Asthma Test (CARAT) (0, 'no control' - 30, 'very high control'). RESULTS: Out of 643 patients with AR, 500 (78%) had asthma as a comorbidity, and 54% had moderate-severe intermittent AR, followed by moderate-severe persistent AR (34%). Compared to the patients with AR alone, patients with AR + asthma had significantly higher RHINASTHMA (e.g., median RHINASTHMA-total score 48.5 vs. 84, respectively) and a significantly lower CARAT score (median CARAT-total score 23 vs. 16.5, respectively). Upon stratifying asthma based on severity, AR patients with severe persistent asthma had worse HRQoL and control than those with mild persistent asthma. The association was significantly higher among non-obese participants compared to obese ones, with RHINASTHMA-upper symptoms score but not with CARAT. CONCLUSIONS: Our observation of poorer HRQoL and symptoms control in AR patients with comorbid asthma supports the importance of a comprehensive approach for the management of AR in case of a comorbid allergic condition.

Appropriateness of psychopharmacological therapies to psychiatric diagnoses in persons with autism spectrum disorder with or without intellectual disabilities: a cross-sectional analytic study.

BACKGROUND: Observational studies highlighted high rates of psychotropic medication in persons with autistic spectrum disorder (ASD) with or without intellectual disability, which seems to be associated with the management of problem behaviors more than co-occurrent psychiatric disorders. The purpose of the study is to investigate psychopharmacology use and diagnoses of co-occurrent psychiatric disorder (PD) in persons with ASD attending a public mental health service in Emilia Romagna, Italy. METHODS: The present study is a multicenter, cross-sectional study. RESULTS: 275 persons out of 486 (56.5%) resulted to receive at least one psychotropic drug, compared to 74 persons (15.2%) that were diagnosed with a PD. 63.6% were on poly-pharmacotherapy (2-10 compounds), with 37.8% receiving 3 or more medications. Antipsychotics were the most frequently prescribed class of psychotropic drugs (89%), followed by antiepileptics/mood stabilizers/lithium (42.1%) and anxiolytics (BDZ) (38.5%). Most common psychiatric disorders were psychotic disorders (29.7%), followed by anxiety disorders (17.5%), bipolar disorders (12.2%), and depressive disorders (9.4%). CONCLUSIONS: Our findings support earlier research showing that many individuals with ASD receive pharmacotherapy without being diagnosed with a co-occurring psychiatric disorder, indicating that the main reasons for prescription and the type of compound frequently have little to no link with specific psychopathology.

Niosomes as Biocompatible Scaffolds for the Multivalent Presentation of Tumor-Associated Antigens (TACAs) to the Immune System.

Fully synthetic tumor-associated carbohydrate antigen (TACA)-based vaccines are a promising strategy to treat cancer. To overcome the intrinsic low immunogenicity of TACAs, the choice of the antigens' analogues and multivalent presentation have been proved to be successful. Here, we present the preparation, characterization, and in vitro screening of niosomes displaying multiple copies of the mucin antigen TnThr (niosomes-7) or of TnThr mimetic 1 (niosomes-2). Unprecedentedly, structural differences, likely related to the carbohydrate portions, were observed for the two colloidal systems. Both niosomal systems are stable, nontoxic and endowed with promising immunogenic properties.

The dynamical Matryoshka model: 2. Modeling of local lipid dynamics at the sub-nanosecond timescale in phospholipid membranes.

Biological membranes are generally formed by lipids and proteins. Often, the membrane properties are studied through model membranes formed by phospholipids only. They are molecules composed by a hydrophilic head group and hydrophobic tails, which can present a panoply of various motions, including small localized movements of a few atoms up to the diffusion of the whole lipid or collective motions of many of them. In the past, efforts were made to measure these motions experimentally by incoherent neutron scattering and to quantify them, but with upcoming modern neutron sources and instruments, such models can now be improved. In the present work, we expose a quantitative and exhaustive study of lipid dynamics on DMPC and DMPG membranes, using the Matryoshka model recently developed by our group. The model is confronted here to experimental data collected on two different membrane samples, at three temperatures and two instruments. Despite such complexity, the model describes reliably the data and permits to extract a series of parameters. The results compare also very well to other values found in the literature.

The dynamical Matryoshka model: 3. Diffusive nature of the atomic motions contained in a new dynamical model for deciphering local lipid dynamics.

In accompanying papers [Bicout et al., BioRxiv https://doi.org/10.1101/2021.09.21.461198 (2021); Cissé et al., BioRxiv https://doi.org/10.1101/2022.03.30.486370 (2022)], a new model called Matryoshka model has been proposed to describe the geometry of atomic motions in phospholipid molecules in bilayers and multilamellar vesicles based on their quasielastic neutron scattering (QENS) spectra. Here, in order to characterize the relaxational aspects of this model, the energy widths of the QENS spectra of the samples were analyzed first in a model-free way. The spectra were decomposed into three Lorentzian functions, which are classified as slow, intermediate, and fast motions depending on their widths. The analysis provides the diffusion coefficients, residence times, and geometrical parameters for the three classes of motions. The results corroborate the parameter values such as the amplitudes and the mobile fractions of atomic motions obtained by the application of the Matryoshka model to the same samples. Since the current analysis was carried out independently of the development of the Matryoshka model, the present results enhance the validity of the model. The model will serve as a powerful tool to decipher the dynamics of lipid molecules not only in model systems, but also in more complex systems such as mixtures of different kinds of lipids or natural cell membranes.

Strikingly Different Roles of SARS-CoV-2 Fusion Peptides Uncovered by Neutron Scattering.

Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.

Polystyrene perturbs the structure, dynamics, and mechanical properties of DPPC membranes: An experimental and computational study.

Synthetic plastic oligomers can interact with the cells of living organisms by different ways. They can be intentionally administered to the human body as part of nanosized biomedical devices. They can be inhaled by exposed workers, during the production of multicomponent, polymer-based nanocomposites. They can leak out of food packaging. Most importantly, they can result from the degradation of plastic waste, and enter the food chain. A physicochemical characterization of the effects of synthetic polymers on the structure and dynamics of cell components is still lacking. Here, we combine a wide spectrum of experimental techniques (calorimetry, x-ray, and neutron scattering) with atomistic Molecular Dynamics simulations to study the interactions between short chains of polystyrene (25 monomers) and model lipid membranes (DPPC, in both gel and fluid phase). We find that doping doses of polystyrene oligomers alter the thermal properties of DPPC, stabilizing the fluid lipid phase. They perturb the membrane structure and dynamics, in a concentration-dependent fashion. Eventually, they modify the mechanical properties of DPPC, reducing its bending modulus in the fluid phase. Our results call for a systematic, interdisciplinary assessment of the mechanisms of interaction of synthetic, everyday use polymers with cell membranes.

Smart Device for Biologically Enhanced Functional Regeneration of Osteo-Tendon Interface.

The spontaneous healing of a tendon laceration results in the formation of scar tissue, which has lower functionality than the original tissue. Moreover, chronic non-healing tendon injuries frequently require surgical treatment. Several types of scaffolds have been developed using the tissue engineering approach, to complement surgical procedures and to enhance the healing process at the injured site. In this work, an electrospun hybrid tubular scaffold was designed to mimic tissue fibrous arrangement and extracellular matrix (ECM) composition, and to be extemporaneously loaded into the inner cavity with human platelet lysate (PL), with the aim of leading to complete post-surgery functional regeneration of the tissue for functional regeneration of the osteo-tendon interface. For this purpose, pullulan (P)/chitosan (CH) based polymer solutions were enriched with hydroxyapatite nanoparticles (HP) and electrospun. The nanofibers were collected vertically along the length of the scaffold to mimic the fascicle direction of the tendon tissue. The scaffold obtained showed tendon-like mechanical performance, depending on HP content and tube size. The PL proteins were able to cross the scaffold wall, and in vitro studies have demonstrated that tenocytes and osteoblasts are able to adhere to and proliferate onto the scaffold in the presence of PL; moreover, they were also able to produce either collagen or sialoproteins, respectively-important components of ECM. These results suggest that HP and PL have a synergic effect, endorsing PL-loaded HP-doped aligned tubular scaffolds as an effective strategy to support new tissue formation in tendon-to-bone interface regeneration.

A Structurally Simple Vaccine Candidate Reduces Progression and Dissemination of Triple-Negative Breast Cancer.

The Tn antigen is a well-known tumor-associated carbohydrate determinant, often incorporated in glycopeptides to develop cancer vaccines. Herein, four copies of a conformationally constrained mimetic of the antigen TnThr (GalNAc-Thr) were conjugated to the adjuvant CRM197, a protein licensed for human use. The resulting vaccine candidate, mime[4]CRM elicited a robust immune response in a triple-negative breast cancer mouse model, correlated with high frequency of CD4+ T cells and low frequency of M2-type macrophages, which reduces tumor progression and lung metastasis growth. Mime[4]CRM-mediated activation of human dendritic cells is reported, and the proliferation of mime[4]CRM-specific T cells, in cancer tissue and peripheral blood of patients with breast cancer, is demonstrated. The locked conformation of the TnThr mimetic and a proper presentation on the surface of CRM197 may explain the binding of the conjugate to the anti-Tn antibody Tn218 and its efficacy to fight cancer cells in mice.

The molecular dynamics of bacterial spore and the role of calcium dipicolinate in core properties at the sub-nanosecond time-scale.

Bacterial spores are among the most resistant forms of life on Earth. Their exceptional resistance properties rely on various strategies, among them the core singular structure, organization and hydration. By using elastic incoherent neutron scattering, we probed the dynamics of Bacillus subtilis spores to determine whether core macromolecular motions at the sub-nanosecond timescale could also contribute to their resistance to physical stresses. In addition, in order to better specify the role of the various spore components, we used different mutants lacking essential structure such as the coat (PS4150 mutant), or the calcium dipicolinic acid complex (CaDPA) located in the core (FB122 mutant). PS4150 allows to better probe the core's dynamics, as proteins of the coat represent an important part of spore proteins, and FB122 gives information about the role of the large CaDPA depot for the mobility of core's components. We show that core's macromolecular mobility is not particularly constrained at the sub-nanosecond timescale in spite of its low water content as some dynamical characteristics as force constants are very close to those of vegetative bacteria such as Escherichia coli or to those of fully hydrated proteins. Although the force constants of the coatless mutant are similar to the wild-type's ones, it has lower mean square displacements (MSDs) at high Q showing that core macromolecules are somewhat more constrained than the rest of spore components. However, no behavior reflecting the glassy state regularly evoked in the literature could be drawn from our data. As hydration and macromolecules' mobility are highly correlated, the previous assumption, that core low water content might explain spores' exceptional resistance properties seems unlikely. Thus, we confirm recent theories, suggesting that core water is mostly as free as bulk water and proteins/macromolecules are fully hydrated. The germination of spores leads to a much less stable system with a force constant of 0.1 N/m and MSDs ~2.5 times higher at low Q than in the dormant state. DPA has also an influence on core mobility with a slightly lower force constant for the DPA-less mutant than for the wild-type, and MSDs that are ~ 1.8 times higher on average than for the wild-type at low Q. At high Q, germinated and DPA-less spores were very similar to the wild-type ones, showing that DPA and core compact structure might influence large amplitude motions rather than local dynamics of macromolecules.

A Quasielastic Neutron Scattering Investigation on the Molecular Self-Dynamics of Human Myelin Protein P2.

The human myelin protein P2 is a membrane binding protein believed to maintain correct lipid composition and organization in peripheral nerve myelin. Its function is related to its ability to stack membranes, and this function can be enhanced by the P38G mutation, whereby the overall protein structure does not change but the molecular dynamics increase. Mutations in P2 are linked to human peripheral neuropathy. Here, the dynamics of wild-type P2 and the P38G variant were studied using quasielastic neutron scattering on time scales from 10 ps to 1 ns at 300 K. The results suggest that the mutant protein dynamics are increased on both the fastest and the slowest measured time scales, by increasing the dynamics amplitude and/or the portion of atoms participating in the movement.

Evidence of a low-temperature dynamical transition in concentrated microgels.

A low-temperature dynamical transition has been reported in several proteins. We provide the first observation of a "protein-like" dynamical transition in nonbiological aqueous environments. To this aim, we exploit the popular colloidal system of poly-N-isopropylacrylamide (PNIPAM) microgels, extending their investigation to unprecedentedly high concentrations. Owing to the heterogeneous architecture of the microgels, water crystallization is avoided in concentrated samples, allowing us to monitor atomic dynamics at low temperatures. By elastic incoherent neutron scattering and molecular dynamics simulations, we find that a dynamical transition occurs at a temperature T d ~ 250 K, independently from PNIPAM mass fraction. However, the transition is smeared out on approaching dry conditions. The quantitative agreement between experiments and simulations provides evidence that the transition occurs simultaneously for PNIPAM and water dynamics. The similarity of these results with hydrated protein powders suggests that the dynamical transition is a generic feature in complex macromolecular systems, independently from their biological function.

Raman and Infrared spectroscopies and X-ray diffraction data on bupivacaine and ropivacaine complexed with 2-hydroxypropyl-ß-cyclodextrin.

The data presented in this article are related to the research article entitled "Probing the dynamics of complexed local anesthetics via neutron scattering spectroscopy and DFT calculations (http://dx.doi.org/10.1016/j.ijpharm.2017.03.051)" (Martins et al., 2017) [1]. This work shows the molecular and structural behavior of the local anesthetics (LAs) bupivacaine (BVC, C18H28N2O) and ropivacaine (RVC, C17H26N2O) before and after complexation with the water-soluble oligosaccharide 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD).

The Dynamical Transition of Lipid Multilamellar Bilayers as a Matter of Cooperativity.

The present study is the application of a two-state model formerly developed by Bicout and Zaccai [ Bicout , D. J. and Zaccai , G. Biophys. J. 2001 , 80 ( 3 ), 1115 - 1123 ] to describe the dynamical transition exhibited in the atomic mean square displacements of biological samples in terms of dynamic and thermodynamic parameters. Data were obtained by elastic incoherent neutron scattering on 1,2-dimyristoyl-sn-glycero-3-phosphocholine lipid membranes in various hydration states and on one partially per-deuterated lipid membrane. Fitting the data with the model allowed investigating which parts of lipid molecules were mainly involved in the dynamical transition, heads, tails, or both. Clear differences were found between the fully protonated and partially deuterated membranes. These findings shed light on the question of what is the degree of dynamical cooperativity of the atoms during the transition. Whereas the level of hydration does not significantly affect it, as the dry, the intermediate dry, and fully hydrated membranes all undergo a rather broad transition, the transition of the lipid tails is much sharper and sets in at much lower temperature than that of the heads. Therefore, the dynamical cooperativity appears high among the particles in the tails. Moreover, the transition of the lipid tails has to be completed first before the one of the head groups starts.

Probing the dynamics of complexed local anesthetics via neutron scattering spectroscopy and DFT calculations.

Since potential changes in the dynamics and mobility of drugs upon complexation for delivery may affect their ultimate efficacy, we have investigated the dynamics of two local anesthetic molecules, bupivacaine (BVC, C18H28N2O) and ropivacaine (RVC, C17H26N2O), in both their crystalline forms and complexed with water-soluble oligosaccharide 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD). The study was carried out by neutron scattering spectroscopy, along with thermal analysis, and density functional theory computation. Mean square displacements suggest that RVC may be less flexible in crystalline form than BVC, but both molecules exhibit very similar dynamics when confined in HP-ß-CD. The use of vibrational analysis by density functional theory (DFT) made possible the identification of molecular modes that are most affected in both molecules by insertion into HP-ß-CD, namely those of the piperidine rings and methyl groups. Nonetheless, the somewhat greater structure in the vibrational spectrum at room temperature of complexed RVC than that of BVC, suggests that the effects of complexation are more severe for the latter. This unique approach to the molecular level study of encapsulated drugs should lead to deeper understanding of their mobility and the respective release dynamics.