Structuring lipid nanoparticles, DNA, and protein corona into stealth bionanoarchitectures for in vivo gene delivery.

Lipid nanoparticles (LNPs) play a crucial role in addressing genetic disorders, and cancer, and combating pandemics such as COVID-19 and its variants. Yet, the ability of LNPs to effectively encapsulate large-size DNA molecules remains elusive. This is a significant limitation, as the successful delivery of large-size DNA holds immense potential for gene therapy.

PEGylation-Dependent Cell Uptake of Lipid Nanoparticles Revealed by Spatiotemporal Correlation Spectroscopy.

Polyethylene glycol (PEG) is a common surface modification for lipid nanoparticles (LNPs) to improve their stability and in vivo circulation time. However, the impact of PEGylation on LNP cellular uptake remains poorly understood. To tackle this issue, we systematically compared plain and PEGylated LNPs by combining dynamic light scattering, electrophoretic light scattering, and synchrotron small-angle X-ray scattering (SAXS) that unveils a striking similarity in size and core structure but a significant reduction in surface charge.

Optical Nanoscopy of Cytokine-Induced Structural Alterations of the Endoplasmic Reticulum and Golgi Apparatus in Insulin-Secreting Cells.

Pro-inflammatory cytokines play a role in the failure of β cells in type 1 and type 2 diabetes. While existing data from 'omics' experiments allow for some understanding of the molecular mechanisms behind cytokine-induced dysfunction in β cells, no report thus far has provided information on the direct imaging of the β cell landscape with nanoscale resolution following cytokine exposure. In this study, we use Airyscan-based optical super-resolution microscopy of Insulinoma 1E (INS-1E) cells to investigate the structural properties of two subcellular membranous compartments involved in the production, maturation and secretion of insulin-containing granules, the endoplasmic reticulum (ER) and the Golgi apparatus (GA).

Protein corona alleviates adverse biological effects of nanoplastics in breast cancer cells.

Pollution from micro- and nanoplastics (MNPs) has long been a topic of concern due to its potential impact on human health. MNPs can circulate through human blood and, thus far, have been found in the lungs, spleen, stomach, liver, kidneys and even in the brain, placenta, and breast milk. While data are already available on the adverse biological effects of pristine MNPs ( oxidative stress, inflammation, cytotoxicity, and even cancer induction), no report thus far clarified whether the same effects are modulated by the formation of a protein corona around MNPs.

Machine-learning-guided recognition of α and β cells from label-free infrared micrographs of living human islets of Langerhans.

Human islets of Langerhans are composed mostly of glucagon-secreting α cells and insulin-secreting β cells closely intermingled one another. Current methods for identifying α and β cells involve either fixing islets and using immunostaining or disaggregating islets and employing flow cytometry for classifying α and β cells based on their size and autofluorescence. Neither approach, however, allows investigating the dynamic behavior of α and β cells in a living and intact islet.

Mechanistic Insights into the Superior DNA Delivery Efficiency of Multicomponent Lipid Nanoparticles: An In Vitro and In Vivo Study.

Lipid nanoparticles (LNPs) are currently having an increasing impact on nanomedicines as delivery agents, among others, of RNA molecules (e.g., short interfering RNA for the treatment of hereditary diseases or messenger RNA for the development of COVID-19 vaccines).

Single-cell imaging of α and β cell metabolic response to glucose in living human Langerhans islets.

Here we use a combination of two-photon Fluorescence Lifetime Imaging Microscopy (FLIM) of NAD(P)H free/bound ratio in living HIs with post-fixation, immunofluorescence-based, cell-type identification. FLIM allowed to measure variations in the NAD(P)H free/bound ratio induced by glucose; immunofluorescence data allowed to identify single α and β cells; finally, matching of the two datasets allowed to assign metabolic shifts to cell identity. 312 α and 654 β cells from a cohort of 4 healthy donors, 15 total islets, were measured.

Measuring Molecular Diffusion in Dynamic Subcellular Nanostructures by Fast Raster Image Correlation Spectroscopy and 3D Orbital Tracking.

Here we provide demonstration that fast fluorescence fluctuation spectroscopy is a fast and robust approach to extract information on the dynamics of molecules enclosed within subcellular nanostructures (e.g., organelles or vesicles) which are also moving in the complex cellular environment.

Mapping the native interaction surfaces of PREP1 with PBX1 by cross-linking mass-spectrometry and mutagenesis.

Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1. This interaction stabilizes the two proteins and allows their translocation into the nucleus and thus their transcriptional activity. Here, we have combined cross-linking mass-spectrometry and systematic mutagenesis to detail the binding geometry of the PBX1-PREP1 (and PBX1-MEIS1) complexes, under native in vivo conditions.

Increased soluble urokinase plasminogen activator receptor (suPAR) levels in neovascular age-related macular degeneration: a role for inflammation in the pathogenesis of the disease?

Purpose: To evaluate the plasma concentration of the soluble form of the urokinase-type plasminogen activator receptor ((s)uPAR), an established biomarker of chronic inflammation, in patients affected by neovascular age-related macular degeneration.

Methods: Forty consecutive patients affected by age-related macular degeneration and 52 subjects with no history of the disease were included in this case-control study. The two groups of individuals considered for the study were matched for age, sex, and class of medications taken.

Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells.

The urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein that promotes tissue remodeling, tumor cell adhesion, migration and invasion. uPAR mediates degradation of the extracellular matrix through protease recruitment and enhances cell adhesion, migration and signaling through vitronectin binding and interactions with integrins. Full-length uPAR is released from the cell surface, but the mechanism and significance of uPAR shedding remain obscure.

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin.

Lamellipodial tension, not integrin/ligand binding, is the crucial factor to realise integrin activation and cell migration.

The molecular clutch (MC) model proposes that actomyosin-driven force transmission permits integrin-dependent cell migration. To investigate the MC, we introduced diverse talin (TLN) and integrin variants into Flp-In™ T-Rex™ HEK293 cells stably expressing uPAR. Vitronectin variants served as substrate providing uPAR-mediated cell adhesion and optionally integrin binding.

Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR) by surface plasmon resonance and X-ray crystallography.

The urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycolipid-anchored membrane protein often expressed in the microenvironment of invasive solid cancers and high levels are generally associated with poor patient prognosis (Kriegbaum et al., 2011 [1]). uPAR is organized as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).

Stabilizing a flexible interdomain hinge region harboring the SMB binding site drives uPAR into its closed conformation.

The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored membrane protein, which facilitates extracellular matrix remodeling by focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assembly of its three LU (Ly6/uPAR-like) domains is inherently flexible and binding of uPA drives uPAR into its closed conformation, which presents the higher-affinity state for vitronectin thus providing an allosteric regulatory mechanism. Using a new class of epitope-mapped anti-uPAR monoclonal antibodies (mAbs), we now demonstrate that the reciprocal stabilization is indeed also possible.

The interaction between uPAR and vitronectin triggers ligand-independent adhesion signalling by integrins.

The urokinase-type plasminogen activator receptor (uPAR) is a non-integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure-function analyses of uPAR, VN and integrins, we document that uPAR-mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin-matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding.