Biology subjects

Grasso, F.

Publications and source records attributed to Grasso, F..

3 recordsLinked to original sources

bioRxiv2024

STANDARDISED FLOW CYTOMETRIC PROCEDURE FOR DEEP CHARACTERIZATION OF NANOPARTICLES

In recent years, there has been a notable increasing interest surrounding the identification and quantification of nanosized particles, including extracellular vesicles (EVs) and viruses. The challenge posed by the nano-sized dimension of these particles makes precise examination a significant undertaking. Among the different techniques for the accurate study of EVs, Flow cytometry (FCM) stands out as the ideal method. It is characterized by high sensitivity, low time consumption, non-destructive sampling, and high throughput. In this article, we propose the optimization of FCM procedures to identify, quantify, and purify EVs and virus like particles (VLPs). The protocol aims to reduce artifacts and errors in nano-sized particles counting, overall caused by the swarming effect. Different threshold strategies were compared to ensure result specificity. Additionally, the critical parameters to consider when using conventional FCM outside of the common experimental context of use have also been identified. Finally, fluorescent-EVs sorting protocol was also developed with highly reliable results using a conventional cell sorter.

cell biology↗

bioRxiv2022

Parallel clonal and molecular profiling of hematopoietic stem cells using RNA barcoding

Anucleate cells - platelets and erythrocytes - constitute over 95% of all hematopoietic stem cell (HSC) output, but the clonal dynamics of HSC contribution to these lineages remains largely unexplored. Here, we use lentiviral RNA cellular barcoding and transplantation of HSCs, combined with single-cell RNA-seq, for quantitative analysis of clonal behavior with a multi-lineage readout - for the first time including anucleate and nucleate lineages. We demonstrate that most HSCs steadily contribute to hematopoiesis, but acute platelet depletion shifts the output of multipotent HSCs to the exclusive production of platelets, with the additional emergence of new myeloid-biased clones. Our approach therefore enables comprehensive profiling of multi-lineage output and transcriptional heterogeneity of individual HSCs, giving insight into clonal dynamics in both steady state and under physiological stress.

cell biology↗

bioRxiv2021

Persistent Immunogenicity of Integrase Defective Lentiviral Vectors delivering membrane tethered Native-Like HIV-1 Envelope Trimers

Integrase Defective Lentiviral Vectors (IDLVs) represent an attractive vaccine platform for delivering HIV-1 antigens, given their ability to induce specific and persistent immune responses in both mice and non-human primates (NHPs). Recent advances in HIV-1 immunogen design demonstrated that native-like HIV-1 Envelope (Env) trimers that mimic the structure of virion-associated Env induce neutralization breadth in rabbits and macaques. Here, we describe the development of an IDLV-based HIV-1 vaccine expressing either soluble ConSOSL.UFO.664 or membrane-tethered ConSOSL.UFO.750 native-like Env immunogens with enhanced bNAb epitopes exposure. We show that IDLV can be pseudotyped with properly folded membrane-tethered native-like UFO.750 trimers. After a single IDLV injection in BALB/c mice, IDLV-UFO.750 induced a faster humoral kinetic as well as higher levels of anti-Env IgG compared to IDLV-UFO.664. IDLV-UFO.750 vaccinated cynomolgus macaques developed unusually long-lasting anti-Env IgG antibodies, as underlined by their remarkable half-life both after priming and boost with IDLV. After boosting with recombinant ConM SOSIP.v7 protein, two animals developed neutralization activity against the autologous tier 1B ConS virus mediated by V1/V2 and V3 glycan sites responses. By combining the possibility to display stabilized trimeric Env on the vector particles with the ability to induce sustained humoral responses, IDLVs represent an appropriate strategy for delivering rationally designed antigens to progress towards an effective HIV-1 vaccine.

immunology↗