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By using AFM as a nanografting tool, we grafted micrometer-sized DNA platforms into inert alkanethiol SAMs. Tuning the grafting conditions (surface density of grafting lines and scan rate) allowed us to tailor the molecular density of the DNA platforms. Following the nanografting process, AFM was operated in the low perturbative Quantitative Imaging (QI) mode. The analysis of QI AFM images showed the coexistence of molecular domains of different heights, and thus different densities, within the grafted areas, which were not previously reported using contact AFM imaging. Thinner domains corresponded to low-density DNA regions characterized by loosely packed, randomly oriented DNA strands, while thicker domains corresponded to regions with more densely grafted DNA. Grafting with densely spaced and slow scans increased the size of the high-density domains, resulting in an overall increase in patch height. The structure of the grafted DNA was compared to self-assembled DNA, which was assessed through nanoshaving experiments. Exposing the DNA patches to the target sequence produced an increase in the patch height, indicating that hybridization was accomplished. The relative height increase of the DNA patches upon hybridization was higher in the case of lower density patches due to hybridization leading to a larger molecular reorganization. Low density DNA patches were therefore the most suitable for targeting oligonucleotide sequences.
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DNA , Oligonucleotídeos , Microscopia de Força Atômica , DNA/química , Hibridização de Ácido NucleicoRESUMO
We have studied the self-assembly of 22-base oligonucleotides bound by a short alkyl thiol linker (C6-ssDNA) on flat Au films. The self-assembled monolayer (SAM) was modified by addition of a spacer (mercaptohexanol, MCH). Molecular depositions were monitored in situ by spectroscopic ellipsometry (SE). SAMs were characterized in a liquid environment by coupling SE (difference spectra method) with Atomic Force Microscope (AFM) measurements. We exploited the biofilm thickness obtained by AFM nanolithography and imaging to solve the refractive index/thickness correlation in optical measurements on ultrathin molecular layers. The combined SE/AFM analysis provided reliable estimates of the thickness and the refractive index of the biofilm in the NIR region (650-1300 nm) and revealed new aspects of DNA molecular organization: exposure to MCH leads to an increase of both film thickness and refractive index, which points to a reorganization of C6-ssDNA film. We show that the contribution of the thiol/Au interface has to be included in the optical model to obtain a more reliable determination of the refractive index of the biofilm in a liquid. The careful, correlative characterization of the mixed C6-ssDNA/MCH SAM represents a key step towards the optimization of a robust detection scheme based on helix-helix hybridization.
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DNA de Cadeia Simples/química , Ouro/química , Microscopia de Força Atômica , Análise Espectral , Compostos de Sulfidrila/químicaAssuntos
COVID-19 , Humanos , COVID-19/diagnóstico por imagem , Raios X , SARS-CoV-2 , Radiografia , Medicina Interna , Estudos RetrospectivosRESUMO
Glioblastoma (GBM), one of the deadliest brain tumors, accounts for approximately 50% of all primary malignant CNS tumors, therefore novel, highly effective remedies are urgently needed. Boron neutron capture therapy, which has recently repositioned as a promising strategy to treat high-grade gliomas, requires a conspicuous accumulation of boron atoms in the cancer cells. With the aim of selectively deliver sodium borocaptate (BSH, a 12 B atoms-including molecule already employed in the clinics) to GBM cells, we developed novel cell membrane-derived vesicles (CMVs), overcoming the limits of natural extracellular vesicles as drug carriers, while maintaining their inherent homing abilities that make them preferable to fully synthetic nanocarriers. Purified cell membrane fragments, isolated from patient-derived GBM stem-like cell cultures, were used to prepare nanosized CMVs, which retained some membrane proteins specific of the GBM parent cells and were devoid of potentially detrimental genetic material. In vitro tests evidenced the targeting ability of this novel nanosystem and ruled out any cytotoxicity. The CMVs were successfully loaded with BSH, by following two different procedures, i.e. sonication and electroporation, demonstrating their potential applicability in GBM therapy.
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Terapia por Captura de Nêutron de Boro , Neoplasias Encefálicas , Membrana Celular , Glioblastoma , Humanos , Terapia por Captura de Nêutron de Boro/métodos , Glioblastoma/radioterapia , Glioblastoma/patologia , Glioblastoma/terapia , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/patologia , Membrana Celular/metabolismo , Boroidretos/química , Linhagem Celular Tumoral , Portadores de Fármacos/química , Nanopartículas/química , Compostos de SulfidrilaRESUMO
We review the advances obtained by using Atomic Force Microscopy (AFM)-based approaches in the field of cell/tissue mechanics and adhesion, comparing the solutions proposed and critically discussing them. AFM offers a wide range of detectable forces with a high force sensitivity, thus allowing a broad class of biological issues to be addressed. Furthermore, it allows for the accurate control of the probe position during the experiments, providing spatially resolved mechanical maps of the biological samples with subcellular resolution. Nowadays, mechanobiology is recognized as a subject of great relevance in biotechnological and biomedical fields. Focusing on the past decade, we discuss the intriguing issues of cellular mechanosensing, i.e., how cells sense and adapt to their mechanical environment. Next, we examine the relationship between cell mechanical properties and pathological states, focusing on cancer and neurodegenerative diseases. We show how AFM has contributed to the characterization of pathological mechanisms and discuss its role in the development of a new class of diagnostic tools that consider cell mechanics as new tumor biomarkers. Finally, we describe the unique ability of AFM to study cell adhesion, working quantitatively and at the single-cell level. Again, we relate cell adhesion experiments to the study of mechanisms directly or secondarily involved in pathologies.
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The majority of anticancer agents currently used derive from natural sources: plants, frequently the ones employed in traditional medicines, are an abundant source of mono- and diterpenes, polyphenols, and alkaloids that exert antitumor activity through diverse mechanisms. Unfortunately, many of these molecules are affected by poor pharmacokinetics and limited specificity, shortcomings that may be overcome by incorporating them into nanovehicles. Cell-derived nanovesicles have recently risen to prominence, due to their biocompatibility, low immunogenicity and, above all, targeting properties. However, due to difficult scalability, the industrial production of biologically-derived vesicles and consequent application in clinics is difficult. As an efficient alternative, bioinspired vesicles deriving from the hybridization of cell-derived and artificial membranes have been conceived, revealing high flexibility and appropriate drug delivery ability. In this review, the most recent advances in the application of these vesicles to the targeted delivery of anticancer actives obtained from plants are presented, with specific focus on vehicle manufacture and characterization, and effectiveness evaluation performed through in vitro and in vivo assays. The emerging overall outlook appears promising in terms of efficient drug loading and selective targeting of tumor cells, suggesting further engrossing developments in the future.
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Porous transition metal oxides are widely studied as biocompatible materials for the development of prosthetic implants. Resurfacing the oxide to improve the antibacterial properties of the material is still an open issue, as infections remain a major cause of implant failure. We investigated the functionalization of porous titanium oxide obtained by anodic oxidation with amino acids (Leucine) as a first step to couple antimicrobial peptides to the oxide surface. We adopted a two-step molecular deposition process as follows: self-assembly of aminophosphonates to titanium oxide followed by covalent coupling of Fmoc-Leucine to aminophosphonates. Molecular deposition was investigated step-by-step by Atomic Force Microscopy (AFM) and X-ray Photoemission Spectroscopy (XPS). Since the inherent high roughness of porous titanium hampers the analysis of molecular orientation on the surface, we resorted to parallel experiments on flat titanium oxide thin films. AFM nanoshaving experiments on aminophosphonates deposited on flat TiO2 indicate the formation of an aminophosphonate monolayer while angle-resolved XPS analysis gives evidence of the formation of an oriented monolayer exposing the amine groups. The availability of the amine groups at the outer interface of the monolayer was confirmed on both flat and porous substrates by the following successful coupling with Fmoc-Leucine, as indicated by high-resolution XPS analysis.
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Purpose: To compare the efficacy of two surgical techniques used to remove silicone oil (SiO) emulsion tamponade after pars plana vitrectomy: triple air-fluid exchange (AFX) and balanced salt solution lavage (BSSL). Methods: X-ray photoemission spectroscopy measured silicon content of the dry residue of fluid samples taken during AFX and BSSL. Ten patients underwent AFX and five BSSL. Three fluid samples were taken per patient, and the dry residue of 10 drops per sample were analyzed. A fluid sample from a patient who never received SiO tamponade was also analyzed to set a "blank" reference sample. Results: Patients' demographics showed no significant difference. Sample 1 of the two groups contained comparable silicon content while samples 2 and 3 of the AFX group contained significantly more silicon than that of the BSSL group (15.0 ± 0.1 and 12.0 ± 0.9 for the AFX group vs. 10.7 ± 1.4 and 5.2 ± 0.6 for the BSSL group, respectively; P < 0.05). The cumulative amount of silicon in the three successive samples was also significantly higher for the AFX group (42.3 ± 1.6 vs. 32 ± 2; P < 0.0001). The average silicon content ratio of consecutive samples was significantly higher for the AFX group compared to the BSSL group (0.90 ± 0.01 vs. 0.58 ± 0.06; P = 0.006). Conclusions: Triple AFX removed more silicon than triple lavage. The eye wall actively interacts with silicon emulsion retaining silicon content rather than behaving as a neutral container. Translational Relevance: Triple air-fluid exchange removed more silicon than BSS lavage. Neither technique behaved as a well-mixed box dilution, suggesting the eye walls actively retain emulsion and a dynamic equilibrium is established between silicon dispersion and the eye wall surface.
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Silício , Óleos de Silicone , Humanos , Emulsões , Espectroscopia Fotoeletrônica , Raios XRESUMO
Thermal scanning-probe lithography (t-SPL) is a high-resolution nanolithography technique that enables the nanopatterning of thermosensitive materials by means of a heated silicon tip. It does not require alignment markers and gives the possibility to assess the morphology of the sample in a noninvasive way before, during, and after the patterning. In order to exploit t-SPL at its peak performances, the writing process requires applying an electric bias between the scanning hot tip and the sample, thereby restricting its application to conductive, optically opaque, substrates. In this work, we show a t-SPL-based method, enabling the noninvasive high-resolution nanolithography of photonic nanostructures onto optically transparent substrates across a broad-band visible and near-infrared spectral range. This was possible by intercalating an ultrathin transparent conductive oxide film between the dielectric substrate and the sacrificial patterning layer. This way, nanolithography performances comparable with those typically observed on conventional semiconductor substrates are achieved without significant changes of the optical response of the final sample. We validated this innovative nanolithography approach by engineering periodic arrays of plasmonic nanoantennas and showing the capability to tune their plasmonic response over a broad-band visible and near-infrared spectral range. The optical properties of the obtained systems make them promising candidates for the fabrication of hybrid plasmonic metasurfaces supported onto fragile low-dimensional materials, thus enabling a variety of applications in nanophotonics, sensing, and thermoplasmonics.
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The surface properties of drug containers should reduce the adsorption of the drug and avoid packaging surface/drug interactions, especially in the case of biologically-derived products. Here, we developed a multi-technique approach that combined Differential Scanning Calorimetry (DSC), Atomic Force Microscopy (AFM), Contact Angle (CA), Quartz Crystal Microbalance with Dissipation monitoring (QCM-D), and X-ray Photoemission Spectroscopy (XPS) to investigate the interactions of rhNGF on different pharma grade polymeric materials. Polypropylene (PP)/polyethylene (PE) copolymers and PP homopolymers, both as spin-coated films and injected molded samples, were evaluated for their degree of crystallinity and adsorption of protein. Our analyses showed that copolymers are characterized by a lower degree of crystallinity and lower roughness compared to PP homopolymers. In line with this, PP/PE copolymers also show higher contact angle values, indicating a lower surface wettability for the rhNGF solution on copolymers than PP homopolymers. Thus, we demonstrated that the chemical composition of the polymeric material and, in turn, its surface roughness determine the interaction with the protein and identified that copolymers may offer an advantage in terms of protein interaction/adsorption. The combined QCM-D and XPS data indicated that protein adsorption is a self-limiting process that passivates the surface after the deposition of roughly one molecular layer, preventing any further protein adsorption in the long term.
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We present a two-step surface modification process to tailor the micro and nano morphology of niobium oxide layers. Niobium was firstly anodized in spark regime in a Ca- and P-containing solution and subsequently treated by acid etching. The effects of anodizing time and applied potential on the surface morphology is investigated with SEM and AFM, complemented by XPS compositional analysis. Anodizing with a limiting potential of 250 V results in the fast growth of oxide layers with a homogeneous distribution of micro-sized pores. Cracks are, however, observed on 250 V grown layers. Limiting the anodizing potential to 200 V slows down the oxide growth, increasing the anodizing time needed to achieve a uniform pore coverage but produces fracture-free oxide layers. The surface nano morphology is further tuned by a subsequent acid etching process that leads to the formation of nano-sized pits on the anodically grown oxide surface. In vitro tests show that the etching-induced nanostructure effectively promotes cell adhesion and spreading onto the niobium oxide surface.
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Antithrombotic therapy may affect outcomes in major trauma but its role is not fully understood. We aimed to investigate adverse outcomes among those with and without antithrombotic treatment in major trauma. Material and methods: This is a retrospective study conducted at the Emergency Department (ED) of the University Hospital of Genoa, a tertiary trauma center, including all major trauma between January 2019 and December 2020. Adverse outcomes were reviewed among those without antithrombotic treatment (Group 0), on antiplatelet treatment (Group 1), and on anticoagulant treatment (Group 2). Results: We reviewed 349 electronic charts for full analysis. Group 0 were n = 310 (88.8%), Group 1 were n = 26 (7.4%), and Group 2 were n = 13 (3.7%). In-hospital death and ICU admission, respectively, were: n = 16 (5.6%) and n = 81 (26%) in Group 0, none and n = 6 (25%) in Group 1, and n = 2 (15.8%) and n = 4 (30.8%) in Group 2 (p = 0.123-p = 0.874). Altered INR (OR 5.2) and increasing D-dimer levels (AUC: 0.81) correlated to increased mortality. Discussion: Group 2 showed higher mortality than Group 0 and Group 1, however Group 2 had fewer active treatments. Of clotting factors, only altered INR and elevated D-dimer levels were significantly correlated to adverse outcomes. Conclusions: Anticoagulant but not antiplatelet treatment seems to produce the worst outcomes in major trauma.
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Excitons dominate the light absorption and re-emission spectra of monolayer transition-metal dichalcogenides (TMD). Microscopic investigations of the excitonic response in TMD almost invariably extract information from the radiative recombination step, which only constitutes one part of the picture. Here, by exploiting imaging spectroscopic ellipsometry (ISE), we investigate the spatial dependence of the dielectric function of chemical vapor deposition (CVD)-grown WS2 flakes with a microscopic lateral resolution, thus providing information about the spatially varying, exciton-induced light absorption in the monolayer WS2. Comparing the ISE results with imaging photoluminescence spectroscopy data, the presence of several correlated features was observed, along with the unexpected existence of a few uncorrelated characteristics. The latter demonstrates that the exciton-induced absorption and emission features are not always proportional at the microscopic scale. Microstructural modulations across the flakes, having a different influence on the absorption and re-emission of light, are deemed responsible for the effect.
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The morphological and mechanical properties of thiolated ssDNA films self-assembled at different ionic strength on flat gold surfaces have been investigated using Atomic Force Microscopy (AFM). AFM nanoshaving experiments, performed in hard tapping mode, allowed selectively removing molecules from micro-sized regions. To image the shaved areas, in addition to the soft contact mode, we explored the use of the Quantitative Imaging (QI) mode. QI is a less perturbative imaging mode that allows obtaining quantitative information on both sample topography and mechanical properties. AFM analysis showed that DNA SAMs assembled at high ionic strength are thicker and less deformable than films prepared at low ionic strength. In the case of thicker films, the difference between film and substrate Young's moduli could be assessed from the analysis of QI data. The AFM finding of thicker and denser films was confirmed by X-Ray Photoelectron Spectroscopy (XPS) and Spectroscopic Ellipsometry (SE) analysis. SE data allowed detecting the DNA UV absorption on dense monomolecular films. Moreover, feeding the SE analysis with the thickness data obtained by AFM, we could estimate the refractive index of dense DNA films.
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The IEV schedule consisted of epirubicin 100 mg/m2 on day 1, etoposide 150 mg/m2 on days 1-3, and ifosfamide 2.5 g/m2 on days 1-3. Patients who proceeded to haematopoietic stem cell transplants (HDTs) received conditioning therapy with BEAM [for the Hodgkin's Lymphoma (HL) and non-Hodgkin's Lymphoma (NHL) groups], or melphalan 100 mg/m2 and mitoxantrone [for the multiple myeloma (MM) patients]. The study consisted of 65 patients with a median age of 53 years: 27 had aggressive NHL, 20 had HL, 7 had indolent NHL, and 11 had MM. Fifty-five patients received IEV for a disease that was refractory to conventional induction regimens, or that was in first or second relapse; 4 patients were treated with IEV while in complete response (CR) after chemotherapy in order to mobilise peripheral blood stem cells (PBSCs). Ninety percent of patients with HL responded to IEV, and 85% achieved CR. Both aggressive and indolent NHLs were less responsive (ORR 50 and 33%, respectively; CRR 41 and 16.5%, respectively). MM patients displayed an intermediate responsiveness (ORR 50% and CRR 30%). IEV was well tolerated in most patients. No life- threatening infections were recorded. PBSC mobilisation was successful in 37 out of 39 patients (95%) and led to the collection of a median of 16, 12, and 13.7 x 10(6) CD34+ cells/kg in patients with HL, NHL, and MM, respectively. All 37 patients underwent an autologous stem cell transplant following a 1 to 2 month interval after the end of IEV. Two patients were submitted to an allogeneic transplant. The median overall survival rate in HL, aggressive NHL, and indolent NHL is 32 (5-60), 16 (2-46), and 14 (4-42) months, respectively. Median EFS is 31 (5-60), 7 (2-46), and 7.5 (4-42) months, respectively. In conclusion, our study confirms that IEV +/- HDT is a well-tolerated and effective salvage treatment for lymphoid malignancies, and that IEV acts as an excellent stem cell mobiliser.