Small-angle X-ray and neutron scattering applied to lipid-based nanoparticles: Recent advancements across different length scales.

Lipid-based nanoparticles (LNPs), ranging from nanovesicles to non-lamellar assemblies, have gained significant attention in recent years, as versatile carriers for delivering drugs, vaccines, and nutrients. Small-angle scattering methods, employing X-rays (SAXS) or neutrons (SANS), represent unique tools to unveil structure, dynamics, and interactions of such particles on different length scales, spanning from the nano to the molecular scale. This review explores the state-of-the-art on scattering methods applied to unveil the structure of lipid-based nanoparticles and their interactions with drugs and bioactive molecules, to inform their rational design and formulation for medical applications. We will focus on complementary information accessible with X-rays or neutrons, ranging from insights on the structure and colloidal processes at a nanoscale level (SAXS) to details on the lipid organization and molecular interactions of LNPs (SANS). In addition, we will review new opportunities offered by Time-resolved (TR)-SAXS and -SANS for the investigation of dynamic processes involving LNPs. These span from real-time monitoring of LNPs structural evolution in response to endogenous or external stimuli (TR-SANS), to the investigation of the kinetics of lipid diffusion and exchange upon interaction with biomolecules (TR-SANS). Finally, we will spotlight novel combinations of SAXS and SANS with complementary on-line techniques, recently enabled at Large Scale Facilities for X-rays and neutrons. This emerging technology enables synchronized multi-method investigation, offering exciting opportunities for the simultaneous characterization of the structure and chemical or mechanical properties of LNPs.

Pro-inflammatory RNA:DNA Hybrids Are p53 Independently Boosted by Hyperbaric Oxygen: a Subcellular Distribution Analysis by Automated Quantitative Imaging.

PURPOSE: RNA:DNA hybrids are co-transcriptional products with acknowledged cytoplasmic pro-inflammatory role as activators of the cGAS-STING pathway. We recently proved them also as radiation-induced senescence messages for the abscopal effect mediation, demonstrating the need for a functional p53 for their production and release in A549 and H1299 tumour cells. However, little is known about their role under different stress conditions, especially in cancer cells. METHODS: In this work, we open the investigation making use of automated quantitative imaging to characterize the hybrid subcellular distribution in HeLa cells grown under different oxygen pressures or exposed to different ionizing radiation doses. After cell imaging by confocal fluorescent microscopy, we apply automated imaging methods developed on purpose to quantify hybrid foci and nuclear cluster intensity, regional and local density and dimension. RESULTS: We show that alteration of culture oxygenation increases hybrid cytoplasmic presence, especially when caused by an hyperoxic environment, with evident hybrid gathering at the cell membrane. Ionizing radiations always fail to increase hybrids, in accordance with the absence of functional p53 in HeLa cells. However, dose-dependent effects are still evident and suggest a threshold dose of 7.5 Gy for remarkable hybrid reduction. CONCLUSION: Together with our previous results, these data demonstrate for the first time that different types of stress can increase hybrid production in cancer cells and by at least two different pathways, one p53-dependent triggerable by ionizing radiations and one p53-independent triggerable by oxidative stress. Together, our findings provide a starting point for understanding hybrid role in tumour stress response.

Interaction of Metallic Nanoparticles With Biomimetic Lipid Liquid Crystalline Cubic Interfaces.

In the past decades, events occurring at the nano-bio interface (i.e., where engineered nanoparticles (NPs) meet biological interfaces such as biomembranes) have been intensively investigated, to address the cytotoxicity of nanomaterials and boost their clinical translation. In this field, lamellar synthetic model membranes have been instrumental to disentangle non-specific interactions between NPs and planar biological interfaces. Much less is known on nano-biointeractions occurring at highly curved biological interfaces, such as cubic membranes. These non-lamellar architectures play a crucial -but far from understood-role in several biological processes and occur in cells as a defence mechanism against bacterial and viral pathologies, including coronaviruses infections. Despite its relevance, the interaction of cubic membranes with nano-sized objects (such as viral pathogens, biological macromolecules and synthetic NPs) remains largely unexplored to date. Here, we address the interaction of model lipid cubic phase membranes with two prototypical classes of NPs for Nanomedicine, i.e., gold (AuNPs) and silver NPs (AgNPs). To this purpose, we challenged lipid cubic phase membranes, either in the form of dispersed nanoparticles (i.e., cubosomes) or solid-supported layers of nanometric thickness, with citrate-stabilized AuNPs and AgNPs and monitored the interaction combining bulk techniques (UV-visible spectroscopy, Light and Synchrotron Small-Angle X-ray Scattering) with surface methods (Quartz Crystal Microbalance and Confocal Laser Scanning Microscopy). We show that the composition of the metal core of NPs (i.e., Au vs Ag) modulates their adsorption and self-assembly at cubic interfaces, leading to an extensive membrane-induced clustering of AuNPs, while only to a mild adsorption of isolated AgNPs. Such differences mirror opposite effects at the membrane level, where AuNPs induce lipid extraction followed by a fast disruption of the cubic assembly, while AgNPs do not affect the membrane morphology. Finally, we propose an interaction mechanism accounting for the different behaviour of AuNPs and AgNPs at the cubic interface, highlighting a prominent role of NPs' composition and surface chemistry in the overall interaction mechanism.

Co-Density Distribution Maps for Advanced Molecule Colocalization and Co-Distribution Analysis.

Cellular and subcellular spatial colocalization of structures and molecules in biological specimens is an important indicator of their co-compartmentalization and interaction. Presently, colocalization in biomedical images is addressed with visual inspection and quantified by co-occurrence and correlation coefficients. However, such measures alone cannot capture the complexity of the interactions, which does not limit itself to signal intensity. On top of the previously developed density distribution maps (DDMs), here, we present a method for advancing current colocalization analysis by introducing co-density distribution maps (cDDMs), which, uniquely, provide information about molecules absolute and relative position and local abundance. We exemplify the benefits of our method by developing cDDMs-integrated pipelines for the analysis of molecules pairs co-distribution in three different real-case image datasets. First, cDDMs are shown to be indicators of colocalization and degree, able to increase the reliability of correlation coefficients currently used to detect the presence of colocalization. In addition, they provide a simultaneously visual and quantitative support, which opens for new investigation paths and biomedical considerations. Finally, thanks to the coDDMaker software we developed, cDDMs become an enabling tool for the quasi real time monitoring of experiments and a potential improvement for a large number of biomedical studies.

Efficacy of penile low-intensity shockwave treatment for erectile dysfunction: correlation with the severity of cavernous artery disease.

We analyzed the efficacy of penile low-intensity extracorporeal shockwave treatment for erectile dysfunction (ED) combined with cavernous artery disease (CAD). ED was evaluated by the International Index of Erectile Function, subdividing patients into mild and moderate/severe forms. CAD was assessed using penile color Doppler ultrasonography. Patients (n = 111) with a positive outcome after treatment, based upon the minimal clinically important difference of the International Index of ED, were followed up for 3 months and 6 months. We found a significant mean increase in the index of erectile function, with an overall improvement in hemodynamic parameters of the cavernous artery. In particular, 93.9% of the patients with mild ED without CAD responded to treatment and 72.7% resumed normal erectile function. Only 31.2% of the patients with moderate/severe ED and CAD responded to treatment, and none resumed normal erectile function. All patients with mild ED and no CAD maintained the effects of therapy after 3 months, while no patients with moderate/severe ED and CAD maintained the benefits of treatment after 3 months. Thus, patients with mild ED and no CAD have better and longer lasting responses to such treatment, with a higher probability of resuming normal erectile function than patients with moderate/severe ED and CAD.

TP53 drives abscopal effect by secretion of senescence-associated molecular signals in non-small cell lung cancer.

BACKGROUND: Recent developments in abscopal effect strongly support the use of radiotherapy for the treatment of metastatic disease. However, deeper understanding of the molecular mechanisms underlying the abscopal effect are required to best benefit a larger proportion of patients with metastasis. Several groups including ours, reported the involvement of wild-type (wt) p53 in radiation-induced abscopal effects, however very little is known on the role of wtp53 dependent molecular mechanisms. METHODS: We investigated through in vivo and in vitro approaches how wtp53 orchestrates radiation-induced abscopal effects. Wtp53 bearing (A549) and p53-null (H1299) NSCLC lines were xenotransplanted in nude mice, and cultured in 2D monolayers and 3D tumor spheroids. Extracellular vesicles (EVs) were isolated from medium cell culture by ultracentrifugation protocol followed by Nanoparticle Tracking Analysis. Gene expression was evaluated by RT-Real Time, digital qRT-PCR, and dot blot technique. Protein levels were determined by immunohistochemistry, confocal anlysis, western blot techniques, and immunoassay. RESULTS: We demonstrated that single high-dose irradiation (20 Gy) induces significant tumor growth inhibition in contralateral non-irradiated (NIR) A549 xenograft tumors but not in NIR p53-null H1299 or p53-silenced A549 (A549sh/p53) xenografts. We further demonstrates that irradiation of A549 cells in vitro induces a senescence-associated secretory phenotype (SASP) producing extracellular vesicles (EVs) expressing CD63 and carrying DNA:RNA hybrids and LINE-1 retrotransposon. IR-A549 EVs also hamper the colony-forming capability of recipient NIR A549 cells, induce senescent phenotype, nuclear expression of DNA:RNA hybrids, and M1 macrophage polarization. CONCLUSIONS: In our models, we demonstrate that high radiation dose in wtp53 tumors induce the onset of SASP and secretion of CD63+ EVs loaded with DNA:RNA hybrids and LINE-1 retrotransposons that convey senescence messages out of the irradiation field triggering abscopal effect in NIR tumors.

Density Distribution Maps: A Novel Tool for Subcellular Distribution Analysis and Quantitative Biomedical Imaging.

Subcellular spatial location is an essential descriptor of molecules biological function. Presently, super-resolution microscopy techniques enable quantification of subcellular objects distribution in fluorescence images, but they rely on instrumentation, tools and expertise not constituting a default for most of laboratories. We propose a method that allows resolving subcellular structures location by reinforcing each single pixel position with the information from surroundings. Although designed for entry-level laboratory equipment with common resolution powers, our method is independent from imaging device resolution, and thus can benefit also super-resolution microscopy. The approach permits to generate density distribution maps (DDMs) informative of both objects' absolute location and self-relative displacement, thus practically reducing location uncertainty and increasing the accuracy of signal mapping. This work proves the capability of the DDMs to: (a) improve the informativeness of spatial distributions; (b) empower subcellular molecules distributions analysis; (c) extend their applicability beyond mere spatial object mapping. Finally, the possibility of enhancing or even disclosing latent distributions can concretely speed-up routine, large-scale and follow-up experiments, besides representing a benefit for all spatial distribution studies, independently of the image acquisition resolution. DDMaker, a Software endowed with a user-friendly Graphical User Interface (GUI), is also provided to support users in DDMs creation.

Identification of Sclerostin as a Putative New Myokine Involved in the Muscle-to-Bone Crosstalk.

Bone and muscle have been recognized as endocrine organs since they produce and secrete "hormone-like factors" that can mutually influence each other and other tissues, giving rise to a "bone-muscle crosstalk". In our study, we made use of myogenic (C2C12 cells) and osteogenic (2T3 cells) cell lines to investigate the effects of muscle cell-produced factors on the maturation process of osteoblasts. We found that the myogenic medium has inhibitory effects on bone cell differentiation and we identified sclerostin as one of the myokines produced by muscle cells. Sclerostin is a secreted glycoprotein reportedly expressed by bone/cartilage cells and is considered a negative regulator of bone growth due to its role as an antagonist of the Wnt/ß-catenin pathway. Given the inhibitory role of sclerostin in bone, we analyzed its expression by muscle cells and how it affects bone formation and homeostasis. Firstly, we characterized and quantified sclerostin synthesis by a myoblast cell line (C2C12) and by murine primary muscle cells by Western blotting, real-time PCR, immunofluorescence, and ELISA assay. Next, we investigated in vivo production of sclerostin in distinct muscle groups with different metabolic and mechanical loading characteristics. This analysis was done in mice of different ages (6 weeks, 5 and 18 months after birth) and revealed that sclerostin expression is dynamically modulated in a muscle-specific way during the lifespan. Finally, we transiently expressed sclerostin in the hind limb muscles of young mice (2 weeks of age) via in vivo electro-transfer of a plasmid containing the SOST gene in order to investigate the effects of muscle-specific overproduction of the protein. Our data disclosed an inhibitory role of the muscular sclerostin on the bones adjacent to the electroporated muscles. This observation suggests that sclerostin released by skeletal muscle might synergistically interact with osseous sclerostin and potentiate negative regulation of osteogenesis possibly by acting in a paracrine/local fashion. Our data point out a role for muscle as a new source of sclerostin.

Prognostic Value of Ultrasound Stratigraphy in Long-Term Weight Loss: Results from a Nutritional Counseling Program.

BACKGROUND: Weight control through lifestyle interventions represents a suitable strategy to avoid the metabolic, endocrine, and reproductive comorbidities associated with overweight and obesity. Reduced testosterone (T) levels are a worsening factor in overweight males. However, prognostic parameters of long-term weight loss are not readily available. Here, we tested the prognostic value of early variations of anthropometric and hormonal parameters, with a focus on ultrasound stratigraphy (US) and the reduction in body mass index (BMI) associated with nutritional counseling/lifestyle interventions at 6-month follow-up. METHODS: Ninety-five male subjects (BMI 25-34.9 kg/m2) who had undergone nutritional/lifestyle interventions, were retrospectively analyzed for: body weight and composition; US evaluation at the triceps (TRC), abdominal (ABD), and thigh (THI) areas; and circulating levels of T, luteinizing hormone, and follicle-stimulating hormone. Sixty patients (63.2%) completed the 6-month follow-up program. RESULTS: At 6 months, a significant reduction in BMI (26.38 ± 1.55 vs. 31.5 ± 5.0 basal, p < 0.001) and increase in T levels (18 ± 5.4 vs. 9.5 ± 2.3 nmol/L basal, p = 0.04) were observed. Subjects in the highest quartile of the BMI reduction at 6 months (ΔBMI 6 mo), compared to the lowest, showed a significant difference at the 2-month follow-up variation of BMI (p = 0.025), and fat and muscle thickness at the TRC (both p < 0.001) and ABD (p < 0.001 and p = 0.002, respectively) areas. Variation of TRC muscle thickness at 2 months was the only independent predictor of ΔBMI 6 mo in the multiple stepwise regression analysis. CONCLUSIONS: BMI evaluation and US represent useful monitoring tools in the follow-up of nutritional/lifestyle interventions for overweight-to-mildly obese patients. The important effects on motivation and adherence to the intervention program are to be considered.

[Pharmaceutical pottery in the Museum of History of Medicine of Rome "La Sapienza"]. / La ceramica apotecaria nel Museo di Storia della Medicina dell'Università di Roma "La Sapienza".

Chemist's and pharmaceutical vases are a historical, aesthetic, cultural, often careless, patrimony that evokes numerous suggestions. It is not possible to distinguish their history from the history of general ceramics in order to reconstruct the context in which these manufactured object were made. Even though manufacture presents a peculiarity of its own it interlaces with the objects of everyday use and with the production designed to any specific and different uses. Ceramics is one of the most flourishing art in Italy; a revival of its techniques arose just in relation with the 'rebirth' of spices commercial trading in Italy.