Simple fluorinated moiety insertion on Aβ 16-23 peptide for stain-free TEM imaging.

Peptide aggregation and fibre formation are one of the major underlying causes of several neurodegenerative disorders such as Alzheimer's disease. During the past decades the characterisation of these fibres has been widely studied in an attempt to further understand the nature of the related diseases and in an effort to develop treatments. Transmission electron microscopy (TEM) is one of the most commonly used techniques to identify these fibres, but requires the use of a radioactive staining agent. The procedure we report overcomes this drawback through simple addition of a fluorinated moiety to a short Amyloid β sequence via solid phase peptide synthesis (SPPS). This method is synthetically straightforward, widely applicable to different aggregation-prone sequences and, above all, allows for stain-free TEM imaging with improved quality compared to standard imaging procedures. The presence of the fluorinated moiety does not cause major changes in the fibre structure or aggregation, but rather serves to dissipate the microscope's electron beam, thus allowing for high contrast and straightforward imaging by TEM.The authors are grateful for funding from the ERC Starting Investigator grant ASPiRe (no. 240629). The authors are also grateful to Dr Marco Di Antonio for assistance with HPLC purification.This is the final published version of the article. It was originally published in Analyst (Sonzini S, Jones ST, Walsh Z, Scherman OA, Analyst, 2015, 140, 2735, doi:10.1039/c4an02278e) http://dx.doi.org/10.1039/c4an02278

Understanding Intracellular Biology to Improve mRNA Delivery by Lipid Nanoparticles

Poor understanding of intracellular delivery and targeting hinders development of nucleic acid‐based therapeutics transported by nanoparticles. Utilizing a siRNA‐targeting and small molecule profiling approach with advanced imaging and machine learning biological insights is generated into the mechanism of lipid nanoparticle (MC3‐LNP) delivery of mRNA. This workflow is termed Advanced Cellular and Endocytic profiling for Intracellular Delivery (ACE‐ID). A cell‐based imaging assay and perturbation of 178 targets relevant to intracellular trafficking is used to identify corresponding effects on functional mRNA delivery. Targets improving delivery are analyzed by extracting data‐rich phenotypic fingerprints from images using advanced image analysis algorithms. Machine learning is used to determine key features correlating with enhanced delivery, identifying fluid‐phase endocytosis as a productive cellular entry route. With this new knowledge, MC3‐LNP is re‐engineered to target macropinocytosis, and this significantly improves mRNA delivery in vitro and in vivo. The ACE‐ID approach can be broadly applicable for optimizing nanomedicine‐based intracellular delivery systems and has the potential to accelerate the development of delivery systems for nucleic acid‐based therapeutics

Research data supporting "Decreasing amyloid toxicity through an increased rate of aggregation"

Raw data supporting the publication in PCCP. Within the main folder there are more folders each containing data for a specific experimental technique. The data are a collection of experimental data for Circular Dichroism, Thioflavin T fluorescence emission, Dynamic Light Scattering, Isothermal Titration Calorimetry, Transmission Electron Microscopy, Atomic Force Microscopy. In addition, fluorescence microscopy and cell assays data are reported as well.ERC [Starting Investigator grant ASPiRe 240629], EPSRC [EP/G060649/1

Raw data for "Turning Cucurbit[8]uril into a Supramolecular Nanoreactor for Asymmetric Catalysis"

These are all the raw data supporting both the results in the manuscript and in the supporting information. A list of abbreviations used in the files is given within the folder.This work was supported by the EPSRC, NIH, European Research Council Starting GrantsASPiRe 240629 (OAS) and NUCLEOPOLY240080 (AH) and STREP (project MICREAGENTS), the Netherlands Organization for Scientific Research (NWO-Vici Grant) and the Zernike Institute for Advanced Materials

Interaction of a macrocycle with an aggregation-prone region of a monoclonal antibody

Colloidal stability is among the key challenges the pharmaceutical industry faces during the production and manufacturing of protein therapeutics. Self-association and aggregation processes can not only impair therapeutic efficacy but also induce immunogenic responses in patients. Aggregation-prone regions (APRs) consisting of hydrophobic patches are commonly identified as the source for colloidal instability, and rational strategies to mitigate aggregation propensity often require genetic engineering to eliminate hydrophobic amino acid residues. Here, we investigate cucurbit[7]­uril (CB[7]), a water-soluble macrocycle able to form host–guest complexes with aromatic amino acid residues, as a potential excipient to mitigate protein aggregation propensity. Two monoclonal antibodies (mAbs), one harboring an APR and one lacking an APR, were first assessed for their colloidal stability (measured as the translational diffusion coefficient) in the presence and absence of CB[7] using dynamic light scattering. Due to the presence of a tryptophan residue within the APR, we were able to monitor changes in intrinsic fluorescence in response to increasing concentrations of CB[7]. Isothermal titration calorimetry and NMR spectroscopy were then used to characterize the putative host–guest interaction. Our results suggest a stabilizing effect of CB[7] on the aggregation-prone mAb, due to the specific interaction of CB[7] with aromatic amino acid residues located within the APR. This provides a starting point for exploring CB[7] as a candidate excipient for the formulation of aggregation-prone mAbs

Memorie di carta

Si raccolgono in volume otto contributi di sette autrici, uno dei quali scritto a più mani, incentrati su specifici nuclei librari e documentari, raccolti e conservati in vari luoghi d’Italia (dal Trentino Alto Adige alla Liguria, dalla Campania alla Sicilia), in alcuni casi andati dispersi e ricostruiti virtualmente. Le autrici affrontano questioni legate ai temi del movimento, degli acquisti, delle donazioni, della dispersione, della manifattura e della conservazione di manoscritti e libri antichi a stampa, anche in tempi a noi abbastanza vicini

Research data supporting "Selective, non-covalent conjugation of synthetic peptides with recombinant proteins mediated by host–guest chemistry"

Raw data to support experiments reported in the main manuscript and ESI of the publicationThis research data supports “Selective, non-covalent conjugation of synthetic peptides with recombinant proteins mediated by host–guest chemistry” which has been published in “Chemical Communications".This work was supported by the ERC [grant number ASPiRe No. 240629]

Raw data for "Turning Cucurbit[8]uril into a Supramolecular Nanoreactor for Asymmetric Catalysis"

These are all the raw data supporting both the results in the manuscript and in the supporting information. A list of abbreviations used in the files is given within the folder