Synthetic nanopores and nanoparticles for the detection and the manipulation of biological molecules.

2006/2007In this work I present a novel approach to the analysis of biomolecules, and a study on two derived practical applications to evaluate its constraints, limits, and potential benefits, namely a biosensing device and a selective transport through membrane. The new approach is based on a 100-800 nm pore etched in a silicon nitride membrane. A linear target molecule, such as DNA, is inserted in the pore and linked at both termini with anchors, one on each side of the pore. Since the complex is stable and the linked objects have a size that is much larger than the target molecule, manipulation, pore closure/opening, possible interactions, stretching and other forces, and in general several characteristics and behaviours of the molecules can be studied at the pore interface. The realization of such a device is preliminary to the development of novel pore-based analytical tools. The principle was applied for the development of a biosensing device. Biosensing devices that perform electrical signal detection are facing the need of being both extremely small and highly sensitive, that is particularly challenging for conventional biosensors where the signal produced is proportional to the surface detecting area. Here, I report the production of a sensor device based on DNA specific displacement of a stable blockade in a synthetic pore section, due to objects associated with the interacting molecules. Thus the signal is proportional to the pore size and not to the surface containing the target/probe molecule. First, I report the setting up of the single components of the device: a complex made of a DNA linker and two particles –the anchors-, the synthetic nanopored membrane and an electrophoretic cell together with an electromagnet -the sensing tools-. Then I show the results of trans-membrane interactions between the objects both outside and inside the sensor device. The applications results related to the biosensor operation are then shown, reporting the detection of the hybridization or the strand-displacement between probes and targets DNA molecules. Finally, I show the operation of a trans-membrane transporter mediated by particles carriers, where the system is exploited to capture and import target molecules through the membrane.XIX Ciclo197

Real-Life Analysis with Erenumab: First Target Therapy in the Episodic and Chronic Migraine’s Prophylaxis

open7noBackground: to research retrospectively the efficacy of Erenumab’s treatment, thus allowing to describe a summary more in line with the reality observed every day in clinical practice, relative to a sample of patients widely heterogeneous. The study aims to confirm the efficacy of Erenumab, in terms of reduction of migraine days per month, from baseline to month 12 of treatment. Additional objectives included a reduction in the number of days of symptomatic drug use and change from baseline in the Migraine Disability Assessment Score Questionnaire (MIDAS); Methods: the analysis included all patients treated for 12 months with Erenumab during the year 2019–2020. The population analyzed consists of twenty-six patients from the Neurology outpatient clinic in Fossombrone. Several quantitative and qualitative variables were recorded by reading the medical records of the patients. The MIDAS was administered to patients to assess the disability related to migraine; Results: at the end of treatment, a statistically significant reduction in the mean number of monthly migraine days, acute medication use per month, and MIDAS questionnaire score was observed; Conclusions: as a preventive treatment of episodic and chronic migraine, our analysis data confirm the efficacy of Erenumab for the prevention of the migraine. The success is achieved in 96% of casesopenMaraia, Zaira; Ricci, Diletta; Rocchi, Marco Bruno Luigi; Moretti, Alessandro; Bufarini, Celestino; Cavaliere, Arturo; Peverini, ManolaMaraia, Zaira; Ricci, Diletta; Rocchi, Marco Bruno Luigi; Moretti, Alessandro; Bufarini, Celestino; Cavaliere, Arturo; Peverini, Manol

The structure of DNA by direct imaging

The structure of DNA was determined in 1953 by x-ray fiber diffraction. Several attempts have been made to obtain a direct image of DNA with alternative techniques. The direct image is intended to allow a quantitative evaluation of all relevant characteristic lengths present in a molecule. A direct image of DNA, which is different from diffraction in the reciprocal space, is difficult to obtain for two main reasons: the intrinsic very low contrast of the elements that form the molecule and the difficulty of preparing the sample while preserving its pristine shape and size. We show that through a preparation procedure compatible with the DNA physiological conditions, a direct image of a single suspended DNA molecule can be obtained. In the image, all relevant lengths of A-form DNA are measurable. A high-resolution transmission electron microscope that operates at 80 keV with an ultimate resolution of 1.5 Å was used for this experiment. Direct imaging of a single molecule can be used as a method to address biological problems that require knowledge at the single-molecule level, given that the average information obtained by x-ray diffraction of crystals or fibers is not sufficient for detailed structure determination, or when crystals cannot be obtained from biological molecules or are not sufficient in understanding multiple protein configurations

Towards a new framework to assess agri-food value chains ' sustainability : the case of chestnut value chain

Abstract: Food systems rely on natural resources for production causing their depletion. Sustainability assessment can encourage farms and agri-food companies to improve sustainability performances. Sustainability assessment frameworks and tools differ in their purposes, scope, methods of application, and required time for execution; however, most of them do not fit with value chains, or they do not cover all sustainability dimensions. Our objective is to propose a holistic framework to assess sustainability at agri-food value chains level. The proposed framework combines the Sustainability Assessment of Food and Agriculture systems (SAFA) (El Hage, 2012) [1] and The Agri-food Evaluation Framework (TEEB) [2]. It incorporates the concepts of Socio-Ecological Systems, Assemblage, and Social Practices. It integrates system dynamics by emphasising human and natural capital stocks and their users. We explain in detail the methodological steps we followed to construct and to apply this new framework to two case studies in Italy and France. The new framework was applied to real-life case studies and has shown its effectiveness and demonstrates its potential for widespread use in similar scenarios

Visualization of single proteins from stripped native cell membranes: a protocol for high-resolution atomic force microscopy

Atomic force microscopy (AFM) proved to be able to obtain high‐resolution three‐dimensional images of single‐membrane proteins, isolated, crystallized, or included in reconstructed model membranes. The extension of this technique to native systems, such as the protein immersed in a cell membrane, needs a careful manipulation of the biological sample to meet the experimental constraints for high‐resolution AFM imaging. In this article, a general protocol for sample preparation is presented, based on the mechanical stretch of the cell membrane. The effectiveness for AFM imaging has been tested on the basis of an integrated optical and AFM approach and the proposed method has been applied to cells expressing cystic fibrosis transmembrane conductance regulator, a channel involved in cystic fibrosis, showing the possibility to identify and analyze single proteins in the plasma membrane

Supramolecular Biopolymers for Tissue Engineering

Supramolecular biopolymers (SBPs) are those polymeric units derived from macromolecules that can assemble with each other by noncovalent interactions. Macromolecular structures are commonly found in living systems such as proteins, DNA/RNA, and polysaccharides. Bioorganic chemistry allows the generation of sequence-specific supramolecular units like SBPs that can be tailored for novel applications in tissue engineering (TE). SBPs hold advantages over other conventional polymers previously used for TE; these materials can be easily functionalized; they are self-healing, biodegradable, stimuli-responsive, and nonimmunogenic. These characteristics are vital for the further development of current trends in TE, such as the use of pluripotent cells for organoid generation, cell-free scaffolds for tissue regeneration, patient-derived organ models, and controlled delivery systems of small molecules. In this review, we will analyse the 3 subtypes of SBPs: peptide-, nucleic acid-, and oligosaccharide-derived. Then, we will discuss the role that SBPs will be playing in TE as dynamic scaffolds, therapeutic scaffolds, and bioinks. Finally, we will describe possible outlooks of SBPs for TE

You Talking to Me? Says the Enteric Nervous System (ENS) to the Microbe. How Intestinal Microbes Interact with the ENS

Mammalian organisms form intimate interfaces with commensal and pathogenic gut microorganisms. Increasing evidence suggests a close interaction between gut microorganisms and the enteric nervous system (ENS), as the first interface to the central nervous system. Each microorganism can exert a different effect on the ENS, including phenotypical neuronal changes or the induction of chemical transmitters that interact with ENS neurons. Some pathogenic bacteria take advantage of the ENS to create a more suitable environment for their growth or to promote the effects of their toxins. In addition, some commensal bacteria can affect the central nervous system (CNS) by locally interacting with the ENS. From the current knowledge emerges an interesting field that may shape future concepts on the pathogen\u2013host synergic interaction. The aim of this narrative review is to report the current findings regarding the inter-relationships between bacteria, viruses, and parasites and the ENS

Direct imaging of DNA fibers: The visage of double helix

Direct imaging becomes important when the knowledge at few/single molecule level is requested and where the diffraction does not allow to get structural and functional information. Here we report on the direct imaging of double stranded (ds) λ-DNA in the A conformation, obtained by combining a novel sample preparation method based on super hydrophobic DNA molecules self-aggregation process with transmission electron microscopy (TEM). The experimental breakthrough is the production of robust and highly ordered paired DNA nanofibers that allowed its direct TEM imaging and the double helix structure revealing

Clustering of Major Histocompatibility Complex-Class I Molecules in Healthy and Cancer Colon Cells Revealed from Their Nanomechanical Properties

The activation of the T cell mediated immune response relies on the fine interaction between the T cell receptor on the immune cell and the antigen-presenting major histocompatibility complex (MHC) molecules on the membrane surface of antigen-presenting cells. Both the distribution and quantity of MHC/peptide complexes and their adequate morphological presentation affect the activation of the immune cells. In several types of cancer the immune response is downregulated due to the low expression of MHC-class I (MHC-I) molecules on the cell's surface, and in addition, the mechanical properties of the membrane seem to play a role. Herein, we investigate the distribution of MHC-I molecules and the related nanoscale mechanical environment on the cell surface of two cell lines derived from colon adenocarcinoma and a healthy epithelial colon reference cell line. Atomic force microscopy (AFM) force spectroscopy analysis using an antibody-tagged pyramidal probe specific for MHC-I molecules and a formula that relates the elasticity of the cell to the energy of adhesion revealed the different population distributions of MHC-I molecules in healthy cells compared to cancer cells. We found that MHC-I molecules are significantly less expressed in cancer cells. Moreover, the local elastic modulus is significantly reduced in cancer cells. We speculate that these results might be related to the proven ability of cancer cells to evade the immune system, not only by reducing MHC-I cell surface expression but also by modifying the local mechanical properties affecting the overall morphology of MHC-I synapse presentation to immune cells

Direct Imaging of DNA Fibers: The Visage of Double Helix

Direct imaging becomes important when the knowledge at few/single molecule level is requested and where the diffraction does not allow to get structural and functional information. Here we report on the direct imaging of double stranded (ds) λ-DNA in the A conformation, obtained by combining a novel sample preparation method based on super hydrophobic DNA molecules self-aggregation process with transmission electron microscopy (TEM). The experimental breakthrough is the production of robust and highly ordered paired DNA nanofibers that allowed its direct TEM imaging and the double helix structure revealing