12 research outputs found
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 acidâbased therapeutics transported by nanoparticles. Utilizing a siRNAâtargeting and small molecule profiling approach with advanced imaging and machine learning biological insights is generated into the mechanism of lipid nanoparticle (MC3âLNP) delivery of mRNA. This workflow is termed Advanced Cellular and Endocytic profiling for Intracellular Delivery (ACEâID). A cellâbased 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 dataârich phenotypic fingerprints from images using advanced image analysis algorithms. Machine learning is used to determine key features correlating with enhanced delivery, identifying fluidâphase endocytosis as a productive cellular entry route. With this new knowledge, MC3âLNP is reâengineered to target macropinocytosis, and this significantly improves mRNA delivery in vitro and in vivo. The ACEâID approach can be broadly applicable for optimizing nanomedicineâbased intracellular delivery systems and has the potential to accelerate the development of delivery systems for nucleic acidâbased therapeutics
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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]Âuril (CB[7]), a water-soluble macrocycle
able to form hostâguest 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 hostâguest 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 piĂš mani, incentrati su specifici nuclei librari e documentari, raccolti e conservati in vari luoghi dâItalia (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
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Research data supporting "Selective, non-covalent conjugation of synthetic peptides with recombinant proteins mediated by hostâguest chemistry"
Raw data to support experiments reported in the main manuscript and ESI of the publicationThis research data supports âSelective, non-covalent conjugation of synthetic peptides with recombinant proteins mediated by hostâguest chemistryâ which has been published in âChemical 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